- Email: [email protected]
[16] Laser capture microdissection in pathology
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ATP~ HK~ADP Glucose
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and G6PdH concentrations are doubled) is added to each sample and the solution incubated for 60 rain at 38 °. Cycling is terminated with 0.5/zl 1 M NaOH and heating to 80 ° for 20 min. A 5-/zl aliquot is transferred to 1 ml 6-PG indicator reagent and NADPH fluorescence is determined.
[16] Laser Capture Microdissection in Pathology By FALKO FEND,
KATJA SPECHT, MARCUS KREMER,
and LETICIAQUINTANILLA-MARTfNEZ Introduction The molecular genetic analysis of pathologically altered tissues has greatly increased our understanding of the etiologies and pathogenesis of human disease processes. The identification of recurrent genetic alterations has a major impact on the pathologic diagnosis of cancer, and conventional morphological tumor classification will rapidly be replaced by defining disease entities based on the integration of clinical, morphological, phenotypical, and genetic information. Moreover, the establishment of individual molecular profiles of tumors may help to identify targets for specific therapeutic intervention. However, primary tissues are a complex mixture of various cell types, and tumors contain an abundance of reactive stromal and inflammatory cells, which frequently outnumber the neoplastic population. This inherent complexity can crucially influence the results of molecular genetic examinations of primary tissues, since many alterations such as loss of heterozygosity or point mutations in tumor suppressor genes or oncogenes can go undetected by standard detection methods if the percentage of "contaminating" stromal cells reaches a certain threshold. In expression profiling of bulk tissue, admixed cell populations can potentiaUy obscure tumor-specific signatures and can make message assignment to specific cell types impossible. Furthermore, early pathologic lesions, such as dysplasia or carcinoma in situ, are frequently inaccessible for conventional molecular analysis.
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To circumvent these problems and to obtain more homogeneous cell populations for molecular analysis, manual and micromanipulator-based microdissection techniques have been developed during the past decade. 1-5 Despite the unquestionable progress achieved with these approaches, such as the unveiling of the lineage and clonality of the malignant cells in Hodgkin's disease, 6 the time-consuming nature of microdissection and the significant manual dexterity required for it have until recently prevented its broad application in pathology. The development of easy-to-handle, laser-assisted technologies such as laser capture microdissection (LCM) or laser microbeam microdissection (LMM) allows rapid and highly precise procurement of purified cell populations suitable for a variety of downstream analyses.7-11 For many diagnostic applications, the use of microdissected cells as template source requires few, if any, modifications of standard protocols, and LCM can easily be integrated into the routines of a molecular pathology laboratory. This chapter gives an overview of applications for laser-assisted microdissection in pathology, focused on, but not restricted to, LCM. We describe various protocols for tissue preparation, microdissection, and DNA and RNA analysis from microdissected tissues. Tissue Preparation Both fresh frozen tissues and fixed, paraffin-embedded specimens, as well as cytological preparations, can be used for LCM. The major difference between routinely fixed and frozen specimens lies in the amount and quality of nucleic acids and protein which can be isolated from these sources. However, pre-LCM tissue preparation and microdissection itself are also critically influenced by the type of starting material. 1 E d'Amore, J. A. Stribley, T. Ohno, G. Wu, R. S. Wickert, J. Delabie, S. H. Hinrichs, and W. C. Chan, Lab. Invest. 76, 219 (1997). 2 G. Deng, Y. Lu, G. Zlotnikov, A. D. Thor, and H. S. Smith, Science 274, 2057 (1996). 3 R. Kiippers, M. Zhao, M. L. Hansmann, and K. Rajewsky, EMBO J. 12, 4955 (1993). 4 L. Whetsell, G. Maw, N. Nadon, E D. Ringer, and E V. Schaefer, Oncogene 7, 2355 (1992). 5 j. j. Going and R. E Lamb, J. Pathol. 179, 121 (1996). 6 R. Kiippers, K. Rajewsky, M. Zhao, G. Simons, R. Laumann, R. Fischer, and M. L. Hansmann, Proc. Natl. Acad. Sci. U.S.A. 91, 10962 (1994). 7 M. BShm, I. Wieland, K. Schiitze, and H. Rithben, Am. J. Pathol. 151, 63 (1997). 8 R. E Bonner, M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997). 9 M. R. Emmert-Buck, R. E Bonnet, E D. Smith, R. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 10 L. Fink, W. Seeger, L. Ermert, J. Hanze, U. Stahl, E Grimminger, W. Kummer, and R. M. Bohle, Nat. Med. 4, 1329 (1998). II K. Schtitze and G. Lahr, Nat. Biotechnol. 16, 737 (1998).
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Paraffin-Embedded Tissues Paraffin embedding, usually preceded by fixation in neutral, buffered 10% formalin, is still the most widely used method for the processing and conservation of diagnostic pathological specimens. However, cross-linking fixatives, such as formalin, lead to significant fragmentation of nucleic acids and cannot be used for techniques such as Southern or Northern blot analysis, which require large amounts of high molecular weight DNA and RNA, respectively. Nevertheless, microdissected paraffin-embedded tissues can serve as template sources for PCR-based analyses of both DNA and RNA, as long as relatively small amplicon sizes are used. Several groups have made efforts to replace formalin with precipitating fixatives such as ethanol, followed by paraffin embedding for improved preservation of nucleic acids.12' 13 Although this represents a promising step to ensure both excellent morphology and superior quality of biologic macromolecules, formalin-fixed specimens still represent the biggest source of archival material for molecular studies. The preparation of paraffin sections for LCM and subsequent DNA or RNA extraction requires little deviation from standard laboratory procedures. Paraffin sections are cut under precautions against cross contamination between different tissue samples, then mounted on standard or plus-charged glass slides, depending on the subsequent staining procedure. After drying at 60 °, usually overnight, the slides are dewaxed in xylene 2x for 5 min, rehydrated through graded alcohols, and finally immersed in distilled water. If the slides are used for RNA extraction, nuclease-free water (DEPC-treated water) should be employed. Hematoxylin-Eosin Staining. The slides are stained in Mayer' hematoxylin solution (Sigma-Aldrich, Deisenhofen, Germany) for 30 sec to 1 min, followed by blueing solution, 70% ethanol, eosin (5-20 sec), and dehydration through graded alcohols and 2 changes of xylene. The use of fresh, 100% ethanol as the final step before xylene is of great importance, because residual humidity can severely interfere with tissue transfer during LCM. Stained, dehydrated slides should be used for LCM as soon as possible, because prolonged storage may lead to reduced tissue transfer and may also influence the quality of nucleic acids. Stained slides should be stored in the presence of desiccants. Several other staining techniques such as hematoxylin or hemalum only, nuclear fast red, and others have been tested and may give equivalent or superior morphology, depending on the type of tissue. 14 Immunohistochemical Staining for Paraffin Sections. A drawback of laserassisted microdissection is the requirement of dehydrated tissue sections or cell 12 S. M. Goldsworthy, E S. Stockton, C. S. Trempus, J. E Foley, and R. R. Maronpot, Mol. Carcinog. 25, 86 (1999). 13 M. Shibutani, C. Uneyama, K. Miyazaki, K. Toyoda, and M. Hirose, Lab. Invest. 80, 199 (2000). 14 T. Ehrig, S. A. Abdulkadir, S. M. Dintzis, J. Milbrandt, and M. A. Watson, J. Mol. Diagn. 3, 22 (2001).
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preparations without coverslip, which leads to a significant decrease in optical resolution and loss of cytological detail. Although routinely stained sections (e.g., hematoxylin-eosin) are frequently sufficient for target recognition in wellstructured tissues, precise microdissection from tissues lacking easily identifiable architectural features such as lymphoid tissues, inflammatory infiltrates, or diffusely infiltrating neoplasms can be virtually impossible. In practice, the precision of laser-assisted microdissection is more frequently limited by difficulties in recognizing the target cells rather than by the technical specifications of the dissection tool. Immunohistochemical or cytochemical staining techniques can improve precision of LCM by rendering high-contrast targets and further allow separation of morphologically homogeneous cell populations according to phenotypical or functional criteria. Standard immunohistochemical procedures for paraffin-embedded tissues do not interfere with LCM and do not seem to have a major influence on subsequent DNA recovery. 1'15 Our laboratory uses for most part routine staining protocols for diagnostic immunohistochemistry, including appropriate heat-induced antigen retrieval, as determined by the primary antibody used. To prevent detachment of slides during the staining procedure, plus-charged (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) or coated (e.g., with poly-L-lysine) slides should be used, which do not interfere with tissue transfer during LCM if handled properly. For the detection of most primary antibodies, the ABC (avidin-biotin complex) technique is employed, with a biotinylated secondary antibody and horseradish peroxidase-labeled avidin as third step. Diaminobenzidine (DAB) as chromogen is well suited for LCM, since the stained sections can be counterstained with hemalum and dehydrated as above, and the stain does not interfere with PCR. In fact, the color precipitate remains on the membrane after digestion or elution of captured cells and can serve as visual control and documentation of dissection specificity (Fig. l). Since strong staining results may be beneficial for the identification of the targeted cells, standard protocols may be optimized accordingly, including overnight incubation with the primary antibody or increased antibody concentrations. A short immunostaining protocol for frozen sections designed to reduce the exposure to aqueous media is described below. LCM of Paraffin Sections. LCM of paraffin sections, whether routinely stained or immunostained, is usually straightforward, and even archival, stained sections can be used successfully after removal of the coverslip with xylene. However, some problems may be encountered during LCM. 1. Poor visualization of targeted cell population. Depending on fissue architecture and type of staining, poor visualization can severely compromise dissection
15E Fend,L. Quintanilla-Martinez,S. Kumar,M. W. Beaty,L. Blum,L. Sorbara, E. S. Jaffe,and M, Raffeld,Am. J. Pathol. 154, 1857(1999).
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A
B
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FIG. 1. (A) LCM of a paraffin section of a composite non-Hodgkin's lymphoma immunostained for CD5. The neoplastic follicles are CD5 negative (asterisks), whereas the interfollicular neoplastic cells express CD5, in addition to reactive T-cells. The holes left behind by the procedure are clearly visible. (Three-step immunoperoxidase technique, x100.) Amplification of rearranged immunoglobulin heavy chain genes from the microdissected CD5+ and C D 5 - cell populations repeatedly rendered two products of different size and sequence (not shown), confirming the presence of two different clones. IF. Fend, L. Quintanilla-Martinez, S. Kumar, M. W. Beaty, L. Blum, L. Sorbara, E. S. Jaffe, and M. Raffeld, Am. J. Pathol. 154, 1857 (1999)]. (B) Cap surface after proteinase K digestion. Although the cellular material has already been removed, the outlines of the captured cells immunostained for CD5 are clearly visible due to the residual DAB precipitate which remains on the thermoplastic membrane and allows control of dissection specificity.
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specificity. Here are some remedies: Use the diffuser or add a drop of xylene to the slide, which works like a coverslip, and create a roadmap image. After evaporation of the xylene, use this image as guidance. Reduce the time in hematoxylin or change to another stain. For certain tissues, the use of immunostaining may be mandatory. Mounted and stained parallel sections can also aid to "navigate" on the slide used for LCM, but this is only appropriate for tissues with larger, predictable anatomical structures which can be followed through step sections. 16 2. The captured tissue remains on the slide. This is probably the most vexing problem. Frequently, insufficient dehydration is the reason, and reimmersion of the section in absolute ethanol followed by xylene may be a remedy. Another strategy is to incubate the slide in water with 3% glycerol before dehydration, which can be of help for various cell or tissue preparations. 17 Uneven section surfaces, slightly tilted placement of the cap, or repositioning of the cap after dissection with adherent cells on the lower surface can compromise tissue contact and dissection efficiency. DNA Extraction. After control of dissection specificity and, if necessary, removal of nonspecifically adherent cells with a light adhesive, put the cap on a 500-/zl tube which contains 50-100/zl of TE buffer with 400/zg/ml proteinase K. The optimal buffer volume depends on the amount of captured cells. For small numbers of cells, the area containing the captured cells can be cut from the cap surface under the microscope with a sterile blade and directly immersed into 10-20 #1 of proteinase K-containing buffer. Altematively, specially designed caps for small amounts of cells can be used in conjunction with the micro-extraction chamber suitable for small fluid volumes (Arcturus Engineering, Santa Clara, CA). Unless a membrane fragment is directly immersed in the buffer, the tube carrying the cap is inverted and incubated at 55 ° for 4 - 8 hr or overnight. Despite the small amount of cells, complete digestion of cells from paraffin-embedded sources requires at least several hours. It is advisable to control for complete digestion by restaining the cap to detect any remaining cell fragments. Because of the limited amount of cells, proteinase K digestion without subsequent purification steps (e.g., organic extraction) is usually sufficient for standard PCR. After heat inactivation of proteinase K and spinning down of any particulate debris, the supernatant can be used for PCR directly. Determination of B-Cell Clonality by PCR Using Consensus Primers against Framework Three Region (FR3) of Immunoglobulin Heavy Chain Genes (IgH). The determination of B- or T-cell clonality of lymphoid proliferations is one of the 16M. H. Wong,J. R. Saam, T. S. Stappenbeck,C. H. Rexer, and J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 97, 12601 (2001). 17L. Jin, C. A. Thompson,X. Qian, S. J. Kuecker,E. Kulig, and R. V. Lloyd, Lab. Invest. 79, 511 (1999).
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most frequently used diagnostic molecular assays in pathology. Since the percentage of clonal cells must reach a certain threshold of at least 2 - 3 % - - i n reality often closer to 10% of the total cell population--to be reliably detected, microdissection can be used to enrich the cell population in question. Besides Hodgkin's disease, many other lesions such as nodular lymphoid infiltrates in the bone marrow or extranodal locations or composite lymphoma lend themselves to microdissection (Fig. 1). However, the use of microdissected tissue fragments for clonality determination requires rigorous control and careful interpretation of results, since the small amounts of lymphoid cells serving as template for amplification can result in "pseudoclonal" amplification products, which should not be equated with malignancy. The effects of formalin fixation with decrease in template quality and subsequent preferential amplification of some rearrangements over others, due to the use of consensus primers with varying binding affinity, can further aggravate this problem. Whenever possible, a single-step PCR should be used to reduce the possibility of clonal amplification products of uncertain relevance. If larger amounts (hundreds to thousands) of cells can be collected, conventional single-step PCR is sufficient to generate enough PCR product for fragment length analysis, direct sequencing, or cloning. Perform all reactions in duplicate, preferably using cells from different microdissections. If clonal bands are not identical in all reactions, they are likely the result of preferential amplification of rare B cells. The following protocol is currently used in our laboratory 15'18: The reaction volume of 25 /zl contains 0.4/zM/liter of each primer (FR3a and LJH19), 0.2 mM/liter dNTPs, 2 mM/liter MgC12, 1.25 U of Taq polymerase (Amplitaq Gold, PerkinElmer, Weiterstadt, Germany) and 1-5 /zl of template DNA. After initial denaturation at 94 ° for 4 min, 40 cycles of amplification are performed at 94 ° for 1 min, 56 ° for 30 sec, and 72 ° for 30 sec, followed by a final extension step at 72 ° for 10 min. The PCR products can be run on a 3% Metaphor gel (FMC Bioproducts, Rockland, ME) or on a 16% polyacrylamide gel. For computer-assisted fragment length analysis, one of the primers is end-labeled with fluorescein, and amplification is performed under identical conditions. Fluorescein-labeled PCR products are analyzed on a high-resolution polyacrylamide gel using an ABI Prism 377 automated sequencer and Genescan software (PerkinElmer). If only very small numbers of cells are available, such as groups of Hodgkin and Reed-Sternberg cells, a seminested protocol using an internal primer directed against homologous sequences of the IgH joining region genes (VLJH) is necessary. 2° After first-round amplification as described above, another 25 cycles under identical conditions are performed, replacing the LJH primer with the VLJH 18M. Kremer,A. D. Cabras,E Fend, S. Schulz, K. Schwarz,H. Hoefler,and M. Werner,Hum. Pathol. 31, 847 (2000). 19G. H. Segal,T. Jorgensen.A. S. Masih, and R. C. Braylan,Hum. Pathol. 25, 1269 (1994). 20M. Kremer,M. Sandherr,B. Geist,A. D. Cabras,H. HSfler,and E Fend,Mod. Pathol. 14, 91 (2001).
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primer and using 3-5 #1 of the first-round product, both undiluted and 1 : 10 diluted, as template for the second round. The isolation of groups of 30 to 50 RS cells per cap and their joint analysis greatly reduce the work needed for clonality analysis of single cells and allow repeat amplification or investigation of other genes. Since contamination by occasional nonneoplastic lymphocytes is likely with this approach, cloning and sequencing of PCR products obtained from several distinct groups of RS cells originating from different microdissections is necessary to confirm the tumor cell origin of the amplification product. Amplified bands are purified from agarose or polyacrylamide gels with appropriate techniques, ligated into the PCR2.1 vector (TA cloning kit, Invitrogen, Carlsbad, CA), and cloned into INVaF' bacteria. 15,2° Sequencing of several inserts obtained from each PCR product will confirm or disprove the clonal identity of the isolated cells, and a minority of contaminating clones will not interfere with the interpretation. RNA Extraction and Real-Time TaqMan RT-PCR in Formalin-Fixed, ParaffinEmbedded Tissues. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues is generally of poor quality because degradation of the RNA can occur before completion of the formalin fixation process. Moreover, as mentioned above, formalin causes cross-linkage of nucleic acids and proteins and covalently modifies RNA by the addition of monomethylol groups to the bases, making RNA extraction, cDNA synthesis, and quantitation analysis problematic. 21 When performing gene expression analysis in FFPE tissues, it is therefore extremely important (a) to choose an RNA extraction procedure that provides only minimally cross-linked RNA and (b) to select very small target sequences in a range of 60-100 bp for real-time RT-PCR, enabling the detection of fragmented and deg r a d e d R N A . 22,23 Generally, there is a huge number of methods available for RNA extraction; however, in our experience, the most successful method in terms of yield of extractable RNA and suitability of the RNA for real-time RT-PCR analysis involves proteinase K digestion. 22 Briefly, RNA from a small number of microdissected cells (20-10,000 cells) is extracted using a modification of the method described by Rupp and L o c k e r . 24 Microdissected cells are transferred to a 1.5-ml microcentrifuge tube and lysed in 200/zl lysis buffer, containing l0 mmol/liter Tris-HC1 (pH 8.0), 0.1 mmol/L EDTA (pH 8.0), 2% sodium dodecyl sulfate (pH 7.3), and freshly added 500 #g/ml proteinase K. Cells are digested for 12 hr at 60 ° until the tissue is completely solubilized. After heat inactivation of the proteinase K for 5 min at 95 °, the RNA is purified by phenol/chloroform extraction: 1/10 volumes 2 M sodium acetate (pH 4.0 ), 1 volume water-saturated acidic 21 N. Masuda, T. Ohnishi, S. Kawamoto, M. Monden, and K. Okubo, Nucleic Acids Res. 27, 4436 (1999). 22 K. Specht, T. Richter, U. MUller, A. Walch, M. Werner, and H. H6fler, Am. J. Pathol. 158, 419 (2001). 23 A. E. Krafft, B. W. Duncan, K. E. Bijwaard, J. K. Taubenberger, and J. H. Lichy, Mol. Diagn. 2, 217 (1997). 24 G. M. Rupp and J. Locker, BioTechniques 6, 56 (1988).
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phenol, and 1/5 volume chloroform are added to the reaction. After vortexing, the samples are put on ice for 15 min and then centrifuged at 14,000 rpm for 20 rain at 4 °. The upper, aqueous phase containing the RNA is transferred to a new microcentrifuge tube and the RNA is precipitated with 2/zl of 10 mg/ml carrier glycogen and 1 volume isopropanol. After incubation for 2 hr at - 2 0 °, the RNA is pelleted by centrifugation at 14,000 rpm for 20 min. The pellet is washed once with 70% ethanol, dried, and resuspended in 20/zl RNase-free H20. If the primers and probes used for subsequent real-time RT-PCR do not span an intron, the removal of genomic DNA by DNase digestion is necessary at this point. cDNA synthesis reaction is carried out with Superscript II Reverse Transcriptase in a final reaction volume of 20/zl as described in the instruction manual (Superscript Choice system) provided by Life Technologies. One-half of the isolated RNA (10/zl) is used for reverse transcription, while a no RT control reaction should be performed in parallel with the other half of the RNA. RNA is annealed with 250 ng random primers at 25 ° for 10 min and then reverse transcribed with 200 U (1 /zl) Superscript reverse transcriptase in 4 #1 of 5× first-strand buffer [250 mM Tris-HCL (pH 8.3), 375 mM KCL, 15 mM MgCI2], 10 mM dithiothreitol (DTT), 1/zl of 0.5 mM of each dNTP, and 1 /zl of RNase inhibitor (40 U)] for 60 min at 42 °. Typically, 1/10-1/20 of the cDNA reaction is then used for subsequent real-time TaqMan PCR. Frozen Tissues
Although frozen tissue represents a superior source for intact biomolecules compared to FFPE tissues, the morphology of frozen sections frequently is poor and further compromises the precision of microdissection. As mentioned above, IHC is an excellent tool for improving the visualization of the target populations. However, standard IHC protocols require several hours of incubation in aqueous media, which results in a significant loss of RNA through the action of ubiquitous RNases. Therefore, several groups have developed immunostaining protocols suitable for LCM and subsequent gene expression analysis. 17,25,26The following rapid immunostalning protocol can be performed with many different primary antibodies and standard reagents and renders mRNA of good quality, although a loss of mRNA in comparison to rapid routine staining (e.g., hematoxylin-eosin) does still occurY Immuno-LCM o f Frozen Sections. The Quick Staining kit (DAKO Corp., Carpinteria, CA) used in the initial publication is no longer commercially avallableY Alternatively, the procedure can be performed with analogous reagents suitable for quick immunostaining, such as a three-step (strept-)avidin-biotin 25E Fend,M. R. Emmert-Buck,R. Chuaqui,K. Cole,J. Lee,L. A. Liotta,andM. Raffeld,Am. J. Pathol. 154, 61 (1999). 26H. Murakami,L. Liotta,and R, A. Star, Kidney Int. 58, 1346(2000).
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FIG. 2. LCM of a frozen section of Hodgkin's disease immunostained for the CD30 antigen. The holes left behind are clearly visible, as well as the membrane staining of other tumor cells. The arrow denotes granulocytes, which show positivity due to endogenous peroxidase activity. (Rapid three-step immunoperoxidase technique, x 600.)
system with a biotinylated secondary antibody and horseradish peroxidase (HRP) (strept-)avidin complex optimized for high sensitivity (Fig. 2). A useful method is the employment of secondary antibodies coupled to a polymer backbone carrying multiple HRP molecules (EnVision, DAKO), which abolish the necessity for a third incubation step before color development and show increased sensitivity. Furthermore, the system is biotin-free and therefore lacks background staining as a result of endogenous biotin. 27
Staining procedure Frozen sections are mounted on charged slides (Superfrost Plus) and immediately refrozen on dry ice. Sections can be stored at - 8 0 °. The sections are briefly thawed at room temperature and immediately immersed in cold acetone for 1-2 min. Drying of the sections before fixation will severely compromise subsequent tissue capture. Do not stain more than 1-3 sections at one time, because multiple slides will lead to a significantly prolonged incubation time. After evaporation of acetone, the slides are rinsed briefly in buffered PBS or Tfis pH 7.4 and incubated with 70-100/zl primary antibody for 1.5-3 rain. 27 U. K'mmnerer, M. Kapp, A. M. Gassel, T. Richter, C. Tank, J. Dietl, and E Ruck, J. Histochem. Cytochem. 49, 623 (2001).
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The optimal dilution and staining time has to be determined individually. The slide is rinsed briefly with buffer and incubated with the secondary reagent for 2-3 min. If a three-step avidin-biotin system with a biotinylated secondary antibody is used, it is followed by incubation with the streptavidin-HRP complex for 2-3 min. Subsequently, the slide is covered with 100 /zl of freshly prepared 3,3'diaminobenzidine solution for 2-5 min. Then the slide is rinsed in water and briefly counterstained with hematoxylin (20-30 sec) if desired. The sections are dehydrated through graded alcohols (15-30 sec each, including twice 100% ethanol) and xylene (twice for 2 rain each). For all aqueous solutions, the use of pure, RNase treated water is recommended. During the incubation steps, placental RNase inhibitor (PerkinElmer) may be added in a concentration of 200-400 U/ml. RNA Extraction. After LCM, the tubes carying the caps with the captured cells are put on ice. RNA extraction is performed using lysis in guanidinium isothiocyanate followed by extraction with water-saturated phenol and subsequent precipitation with cold isopropanol and glycogen added as carrier (Micro RNA isolation kit, Stratagene, La Jolla, CA). If the presence of contaminating DNA is critical, DNase digestion is mandatory. RNA is dissolved in pure water containing 1 /zl of RNase inhibitor and incubated for 2 hr at 37 ° with 10-20 U of DNase I (Genhunter Corp., Nashville, TN). After reextraction of RNA following the same protocol as above, the RNA is dissolved in pure water, and 1/xl of RNase inhibitor is added. Reverse transcription is performed with 2.5/zmol/liter of random hexamers, 250/zmol/liter of each dNTP, and 100 U of MMLV reverse transcriptase (GenHunter) in a final volume of 20/zl. A mock reaction without RT should be performed in parallel.9,15 Depending on the amount of isolated cells, 20/zl of cDNA is usually sufficient for 10-20 or more single-step PCR reactions, and fragments larger than 500 bp can be amplified successfully. Conclusions The above protocols represent a small selection of fairly simple, easy-to-use techniques which can be readily performed in most molecular pathology laboratories. However, methodical improvements and further technical developments are realized at a rapid pace in all relevant areas, including tissue conservation and pre-LCM preparation, identification, and precise isolation of targeted cells, as well as more sensitive downstream analytical techniques adapted to small amounts of tissue, including high throughput screening methods such as cDNA microarrays or proteomics. Our increasing ability to correlate molecular findings with morphology and phenotype on the microscopic level will have a profound impact on all aspects of pathology.
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ATP~ HK~ADP Glucose
~'Jk'G-6P~6PdH .I#6-PG NADP+ ~
~
NADPH
SCHEME VI. Glucose assay.
and G6PdH concentrations are doubled) is added to each sample and the solution incubated for 60 rain at 38 °. Cycling is terminated with 0.5/zl 1 M NaOH and heating to 80 ° for 20 min. A 5-/zl aliquot is transferred to 1 ml 6-PG indicator reagent and NADPH fluorescence is determined.
[16] Laser Capture Microdissection in Pathology By FALKO FEND,
KATJA SPECHT, MARCUS KREMER,
and LETICIAQUINTANILLA-MARTfNEZ Introduction The molecular genetic analysis of pathologically altered tissues has greatly increased our understanding of the etiologies and pathogenesis of human disease processes. The identification of recurrent genetic alterations has a major impact on the pathologic diagnosis of cancer, and conventional morphological tumor classification will rapidly be replaced by defining disease entities based on the integration of clinical, morphological, phenotypical, and genetic information. Moreover, the establishment of individual molecular profiles of tumors may help to identify targets for specific therapeutic intervention. However, primary tissues are a complex mixture of various cell types, and tumors contain an abundance of reactive stromal and inflammatory cells, which frequently outnumber the neoplastic population. This inherent complexity can crucially influence the results of molecular genetic examinations of primary tissues, since many alterations such as loss of heterozygosity or point mutations in tumor suppressor genes or oncogenes can go undetected by standard detection methods if the percentage of "contaminating" stromal cells reaches a certain threshold. In expression profiling of bulk tissue, admixed cell populations can potentiaUy obscure tumor-specific signatures and can make message assignment to specific cell types impossible. Furthermore, early pathologic lesions, such as dysplasia or carcinoma in situ, are frequently inaccessible for conventional molecular analysis.
METHODSINENZYMOLOGY,VOL.356
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To circumvent these problems and to obtain more homogeneous cell populations for molecular analysis, manual and micromanipulator-based microdissection techniques have been developed during the past decade. 1-5 Despite the unquestionable progress achieved with these approaches, such as the unveiling of the lineage and clonality of the malignant cells in Hodgkin's disease, 6 the time-consuming nature of microdissection and the significant manual dexterity required for it have until recently prevented its broad application in pathology. The development of easy-to-handle, laser-assisted technologies such as laser capture microdissection (LCM) or laser microbeam microdissection (LMM) allows rapid and highly precise procurement of purified cell populations suitable for a variety of downstream analyses.7-11 For many diagnostic applications, the use of microdissected cells as template source requires few, if any, modifications of standard protocols, and LCM can easily be integrated into the routines of a molecular pathology laboratory. This chapter gives an overview of applications for laser-assisted microdissection in pathology, focused on, but not restricted to, LCM. We describe various protocols for tissue preparation, microdissection, and DNA and RNA analysis from microdissected tissues. Tissue Preparation Both fresh frozen tissues and fixed, paraffin-embedded specimens, as well as cytological preparations, can be used for LCM. The major difference between routinely fixed and frozen specimens lies in the amount and quality of nucleic acids and protein which can be isolated from these sources. However, pre-LCM tissue preparation and microdissection itself are also critically influenced by the type of starting material. 1 E d'Amore, J. A. Stribley, T. Ohno, G. Wu, R. S. Wickert, J. Delabie, S. H. Hinrichs, and W. C. Chan, Lab. Invest. 76, 219 (1997). 2 G. Deng, Y. Lu, G. Zlotnikov, A. D. Thor, and H. S. Smith, Science 274, 2057 (1996). 3 R. Kiippers, M. Zhao, M. L. Hansmann, and K. Rajewsky, EMBO J. 12, 4955 (1993). 4 L. Whetsell, G. Maw, N. Nadon, E D. Ringer, and E V. Schaefer, Oncogene 7, 2355 (1992). 5 j. j. Going and R. E Lamb, J. Pathol. 179, 121 (1996). 6 R. Kiippers, K. Rajewsky, M. Zhao, G. Simons, R. Laumann, R. Fischer, and M. L. Hansmann, Proc. Natl. Acad. Sci. U.S.A. 91, 10962 (1994). 7 M. BShm, I. Wieland, K. Schiitze, and H. Rithben, Am. J. Pathol. 151, 63 (1997). 8 R. E Bonner, M. Emmert-Buck, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein, and L. A. Liotta, Science 278, 1481 (1997). 9 M. R. Emmert-Buck, R. E Bonnet, E D. Smith, R. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, and L. A. Liotta, Science 274, 998 (1996). 10 L. Fink, W. Seeger, L. Ermert, J. Hanze, U. Stahl, E Grimminger, W. Kummer, and R. M. Bohle, Nat. Med. 4, 1329 (1998). II K. Schtitze and G. Lahr, Nat. Biotechnol. 16, 737 (1998).
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Paraffin-Embedded Tissues Paraffin embedding, usually preceded by fixation in neutral, buffered 10% formalin, is still the most widely used method for the processing and conservation of diagnostic pathological specimens. However, cross-linking fixatives, such as formalin, lead to significant fragmentation of nucleic acids and cannot be used for techniques such as Southern or Northern blot analysis, which require large amounts of high molecular weight DNA and RNA, respectively. Nevertheless, microdissected paraffin-embedded tissues can serve as template sources for PCR-based analyses of both DNA and RNA, as long as relatively small amplicon sizes are used. Several groups have made efforts to replace formalin with precipitating fixatives such as ethanol, followed by paraffin embedding for improved preservation of nucleic acids.12' 13 Although this represents a promising step to ensure both excellent morphology and superior quality of biologic macromolecules, formalin-fixed specimens still represent the biggest source of archival material for molecular studies. The preparation of paraffin sections for LCM and subsequent DNA or RNA extraction requires little deviation from standard laboratory procedures. Paraffin sections are cut under precautions against cross contamination between different tissue samples, then mounted on standard or plus-charged glass slides, depending on the subsequent staining procedure. After drying at 60 °, usually overnight, the slides are dewaxed in xylene 2x for 5 min, rehydrated through graded alcohols, and finally immersed in distilled water. If the slides are used for RNA extraction, nuclease-free water (DEPC-treated water) should be employed. Hematoxylin-Eosin Staining. The slides are stained in Mayer' hematoxylin solution (Sigma-Aldrich, Deisenhofen, Germany) for 30 sec to 1 min, followed by blueing solution, 70% ethanol, eosin (5-20 sec), and dehydration through graded alcohols and 2 changes of xylene. The use of fresh, 100% ethanol as the final step before xylene is of great importance, because residual humidity can severely interfere with tissue transfer during LCM. Stained, dehydrated slides should be used for LCM as soon as possible, because prolonged storage may lead to reduced tissue transfer and may also influence the quality of nucleic acids. Stained slides should be stored in the presence of desiccants. Several other staining techniques such as hematoxylin or hemalum only, nuclear fast red, and others have been tested and may give equivalent or superior morphology, depending on the type of tissue. 14 Immunohistochemical Staining for Paraffin Sections. A drawback of laserassisted microdissection is the requirement of dehydrated tissue sections or cell 12 S. M. Goldsworthy, E S. Stockton, C. S. Trempus, J. E Foley, and R. R. Maronpot, Mol. Carcinog. 25, 86 (1999). 13 M. Shibutani, C. Uneyama, K. Miyazaki, K. Toyoda, and M. Hirose, Lab. Invest. 80, 199 (2000). 14 T. Ehrig, S. A. Abdulkadir, S. M. Dintzis, J. Milbrandt, and M. A. Watson, J. Mol. Diagn. 3, 22 (2001).
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preparations without coverslip, which leads to a significant decrease in optical resolution and loss of cytological detail. Although routinely stained sections (e.g., hematoxylin-eosin) are frequently sufficient for target recognition in wellstructured tissues, precise microdissection from tissues lacking easily identifiable architectural features such as lymphoid tissues, inflammatory infiltrates, or diffusely infiltrating neoplasms can be virtually impossible. In practice, the precision of laser-assisted microdissection is more frequently limited by difficulties in recognizing the target cells rather than by the technical specifications of the dissection tool. Immunohistochemical or cytochemical staining techniques can improve precision of LCM by rendering high-contrast targets and further allow separation of morphologically homogeneous cell populations according to phenotypical or functional criteria. Standard immunohistochemical procedures for paraffin-embedded tissues do not interfere with LCM and do not seem to have a major influence on subsequent DNA recovery. 1'15 Our laboratory uses for most part routine staining protocols for diagnostic immunohistochemistry, including appropriate heat-induced antigen retrieval, as determined by the primary antibody used. To prevent detachment of slides during the staining procedure, plus-charged (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) or coated (e.g., with poly-L-lysine) slides should be used, which do not interfere with tissue transfer during LCM if handled properly. For the detection of most primary antibodies, the ABC (avidin-biotin complex) technique is employed, with a biotinylated secondary antibody and horseradish peroxidase-labeled avidin as third step. Diaminobenzidine (DAB) as chromogen is well suited for LCM, since the stained sections can be counterstained with hemalum and dehydrated as above, and the stain does not interfere with PCR. In fact, the color precipitate remains on the membrane after digestion or elution of captured cells and can serve as visual control and documentation of dissection specificity (Fig. l). Since strong staining results may be beneficial for the identification of the targeted cells, standard protocols may be optimized accordingly, including overnight incubation with the primary antibody or increased antibody concentrations. A short immunostaining protocol for frozen sections designed to reduce the exposure to aqueous media is described below. LCM of Paraffin Sections. LCM of paraffin sections, whether routinely stained or immunostained, is usually straightforward, and even archival, stained sections can be used successfully after removal of the coverslip with xylene. However, some problems may be encountered during LCM. 1. Poor visualization of targeted cell population. Depending on fissue architecture and type of staining, poor visualization can severely compromise dissection
15E Fend,L. Quintanilla-Martinez,S. Kumar,M. W. Beaty,L. Blum,L. Sorbara, E. S. Jaffe,and M, Raffeld,Am. J. Pathol. 154, 1857(1999).
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A
B
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FIG. 1. (A) LCM of a paraffin section of a composite non-Hodgkin's lymphoma immunostained for CD5. The neoplastic follicles are CD5 negative (asterisks), whereas the interfollicular neoplastic cells express CD5, in addition to reactive T-cells. The holes left behind by the procedure are clearly visible. (Three-step immunoperoxidase technique, x100.) Amplification of rearranged immunoglobulin heavy chain genes from the microdissected CD5+ and C D 5 - cell populations repeatedly rendered two products of different size and sequence (not shown), confirming the presence of two different clones. IF. Fend, L. Quintanilla-Martinez, S. Kumar, M. W. Beaty, L. Blum, L. Sorbara, E. S. Jaffe, and M. Raffeld, Am. J. Pathol. 154, 1857 (1999)]. (B) Cap surface after proteinase K digestion. Although the cellular material has already been removed, the outlines of the captured cells immunostained for CD5 are clearly visible due to the residual DAB precipitate which remains on the thermoplastic membrane and allows control of dissection specificity.
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specificity. Here are some remedies: Use the diffuser or add a drop of xylene to the slide, which works like a coverslip, and create a roadmap image. After evaporation of the xylene, use this image as guidance. Reduce the time in hematoxylin or change to another stain. For certain tissues, the use of immunostaining may be mandatory. Mounted and stained parallel sections can also aid to "navigate" on the slide used for LCM, but this is only appropriate for tissues with larger, predictable anatomical structures which can be followed through step sections. 16 2. The captured tissue remains on the slide. This is probably the most vexing problem. Frequently, insufficient dehydration is the reason, and reimmersion of the section in absolute ethanol followed by xylene may be a remedy. Another strategy is to incubate the slide in water with 3% glycerol before dehydration, which can be of help for various cell or tissue preparations. 17 Uneven section surfaces, slightly tilted placement of the cap, or repositioning of the cap after dissection with adherent cells on the lower surface can compromise tissue contact and dissection efficiency. DNA Extraction. After control of dissection specificity and, if necessary, removal of nonspecifically adherent cells with a light adhesive, put the cap on a 500-/zl tube which contains 50-100/zl of TE buffer with 400/zg/ml proteinase K. The optimal buffer volume depends on the amount of captured cells. For small numbers of cells, the area containing the captured cells can be cut from the cap surface under the microscope with a sterile blade and directly immersed into 10-20 #1 of proteinase K-containing buffer. Altematively, specially designed caps for small amounts of cells can be used in conjunction with the micro-extraction chamber suitable for small fluid volumes (Arcturus Engineering, Santa Clara, CA). Unless a membrane fragment is directly immersed in the buffer, the tube carrying the cap is inverted and incubated at 55 ° for 4 - 8 hr or overnight. Despite the small amount of cells, complete digestion of cells from paraffin-embedded sources requires at least several hours. It is advisable to control for complete digestion by restaining the cap to detect any remaining cell fragments. Because of the limited amount of cells, proteinase K digestion without subsequent purification steps (e.g., organic extraction) is usually sufficient for standard PCR. After heat inactivation of proteinase K and spinning down of any particulate debris, the supernatant can be used for PCR directly. Determination of B-Cell Clonality by PCR Using Consensus Primers against Framework Three Region (FR3) of Immunoglobulin Heavy Chain Genes (IgH). The determination of B- or T-cell clonality of lymphoid proliferations is one of the 16M. H. Wong,J. R. Saam, T. S. Stappenbeck,C. H. Rexer, and J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 97, 12601 (2001). 17L. Jin, C. A. Thompson,X. Qian, S. J. Kuecker,E. Kulig, and R. V. Lloyd, Lab. Invest. 79, 511 (1999).
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most frequently used diagnostic molecular assays in pathology. Since the percentage of clonal cells must reach a certain threshold of at least 2 - 3 % - - i n reality often closer to 10% of the total cell population--to be reliably detected, microdissection can be used to enrich the cell population in question. Besides Hodgkin's disease, many other lesions such as nodular lymphoid infiltrates in the bone marrow or extranodal locations or composite lymphoma lend themselves to microdissection (Fig. 1). However, the use of microdissected tissue fragments for clonality determination requires rigorous control and careful interpretation of results, since the small amounts of lymphoid cells serving as template for amplification can result in "pseudoclonal" amplification products, which should not be equated with malignancy. The effects of formalin fixation with decrease in template quality and subsequent preferential amplification of some rearrangements over others, due to the use of consensus primers with varying binding affinity, can further aggravate this problem. Whenever possible, a single-step PCR should be used to reduce the possibility of clonal amplification products of uncertain relevance. If larger amounts (hundreds to thousands) of cells can be collected, conventional single-step PCR is sufficient to generate enough PCR product for fragment length analysis, direct sequencing, or cloning. Perform all reactions in duplicate, preferably using cells from different microdissections. If clonal bands are not identical in all reactions, they are likely the result of preferential amplification of rare B cells. The following protocol is currently used in our laboratory 15'18: The reaction volume of 25 /zl contains 0.4/zM/liter of each primer (FR3a and LJH19), 0.2 mM/liter dNTPs, 2 mM/liter MgC12, 1.25 U of Taq polymerase (Amplitaq Gold, PerkinElmer, Weiterstadt, Germany) and 1-5 /zl of template DNA. After initial denaturation at 94 ° for 4 min, 40 cycles of amplification are performed at 94 ° for 1 min, 56 ° for 30 sec, and 72 ° for 30 sec, followed by a final extension step at 72 ° for 10 min. The PCR products can be run on a 3% Metaphor gel (FMC Bioproducts, Rockland, ME) or on a 16% polyacrylamide gel. For computer-assisted fragment length analysis, one of the primers is end-labeled with fluorescein, and amplification is performed under identical conditions. Fluorescein-labeled PCR products are analyzed on a high-resolution polyacrylamide gel using an ABI Prism 377 automated sequencer and Genescan software (PerkinElmer). If only very small numbers of cells are available, such as groups of Hodgkin and Reed-Sternberg cells, a seminested protocol using an internal primer directed against homologous sequences of the IgH joining region genes (VLJH) is necessary. 2° After first-round amplification as described above, another 25 cycles under identical conditions are performed, replacing the LJH primer with the VLJH 18M. Kremer,A. D. Cabras,E Fend, S. Schulz, K. Schwarz,H. Hoefler,and M. Werner,Hum. Pathol. 31, 847 (2000). 19G. H. Segal,T. Jorgensen.A. S. Masih, and R. C. Braylan,Hum. Pathol. 25, 1269 (1994). 20M. Kremer,M. Sandherr,B. Geist,A. D. Cabras,H. HSfler,and E Fend,Mod. Pathol. 14, 91 (2001).
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primer and using 3-5 #1 of the first-round product, both undiluted and 1 : 10 diluted, as template for the second round. The isolation of groups of 30 to 50 RS cells per cap and their joint analysis greatly reduce the work needed for clonality analysis of single cells and allow repeat amplification or investigation of other genes. Since contamination by occasional nonneoplastic lymphocytes is likely with this approach, cloning and sequencing of PCR products obtained from several distinct groups of RS cells originating from different microdissections is necessary to confirm the tumor cell origin of the amplification product. Amplified bands are purified from agarose or polyacrylamide gels with appropriate techniques, ligated into the PCR2.1 vector (TA cloning kit, Invitrogen, Carlsbad, CA), and cloned into INVaF' bacteria. 15,2° Sequencing of several inserts obtained from each PCR product will confirm or disprove the clonal identity of the isolated cells, and a minority of contaminating clones will not interfere with the interpretation. RNA Extraction and Real-Time TaqMan RT-PCR in Formalin-Fixed, ParaffinEmbedded Tissues. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues is generally of poor quality because degradation of the RNA can occur before completion of the formalin fixation process. Moreover, as mentioned above, formalin causes cross-linkage of nucleic acids and proteins and covalently modifies RNA by the addition of monomethylol groups to the bases, making RNA extraction, cDNA synthesis, and quantitation analysis problematic. 21 When performing gene expression analysis in FFPE tissues, it is therefore extremely important (a) to choose an RNA extraction procedure that provides only minimally cross-linked RNA and (b) to select very small target sequences in a range of 60-100 bp for real-time RT-PCR, enabling the detection of fragmented and deg r a d e d R N A . 22,23 Generally, there is a huge number of methods available for RNA extraction; however, in our experience, the most successful method in terms of yield of extractable RNA and suitability of the RNA for real-time RT-PCR analysis involves proteinase K digestion. 22 Briefly, RNA from a small number of microdissected cells (20-10,000 cells) is extracted using a modification of the method described by Rupp and L o c k e r . 24 Microdissected cells are transferred to a 1.5-ml microcentrifuge tube and lysed in 200/zl lysis buffer, containing l0 mmol/liter Tris-HC1 (pH 8.0), 0.1 mmol/L EDTA (pH 8.0), 2% sodium dodecyl sulfate (pH 7.3), and freshly added 500 #g/ml proteinase K. Cells are digested for 12 hr at 60 ° until the tissue is completely solubilized. After heat inactivation of the proteinase K for 5 min at 95 °, the RNA is purified by phenol/chloroform extraction: 1/10 volumes 2 M sodium acetate (pH 4.0 ), 1 volume water-saturated acidic 21 N. Masuda, T. Ohnishi, S. Kawamoto, M. Monden, and K. Okubo, Nucleic Acids Res. 27, 4436 (1999). 22 K. Specht, T. Richter, U. MUller, A. Walch, M. Werner, and H. H6fler, Am. J. Pathol. 158, 419 (2001). 23 A. E. Krafft, B. W. Duncan, K. E. Bijwaard, J. K. Taubenberger, and J. H. Lichy, Mol. Diagn. 2, 217 (1997). 24 G. M. Rupp and J. Locker, BioTechniques 6, 56 (1988).
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phenol, and 1/5 volume chloroform are added to the reaction. After vortexing, the samples are put on ice for 15 min and then centrifuged at 14,000 rpm for 20 rain at 4 °. The upper, aqueous phase containing the RNA is transferred to a new microcentrifuge tube and the RNA is precipitated with 2/zl of 10 mg/ml carrier glycogen and 1 volume isopropanol. After incubation for 2 hr at - 2 0 °, the RNA is pelleted by centrifugation at 14,000 rpm for 20 min. The pellet is washed once with 70% ethanol, dried, and resuspended in 20/zl RNase-free H20. If the primers and probes used for subsequent real-time RT-PCR do not span an intron, the removal of genomic DNA by DNase digestion is necessary at this point. cDNA synthesis reaction is carried out with Superscript II Reverse Transcriptase in a final reaction volume of 20/zl as described in the instruction manual (Superscript Choice system) provided by Life Technologies. One-half of the isolated RNA (10/zl) is used for reverse transcription, while a no RT control reaction should be performed in parallel with the other half of the RNA. RNA is annealed with 250 ng random primers at 25 ° for 10 min and then reverse transcribed with 200 U (1 /zl) Superscript reverse transcriptase in 4 #1 of 5× first-strand buffer [250 mM Tris-HCL (pH 8.3), 375 mM KCL, 15 mM MgCI2], 10 mM dithiothreitol (DTT), 1/zl of 0.5 mM of each dNTP, and 1 /zl of RNase inhibitor (40 U)] for 60 min at 42 °. Typically, 1/10-1/20 of the cDNA reaction is then used for subsequent real-time TaqMan PCR. Frozen Tissues
Although frozen tissue represents a superior source for intact biomolecules compared to FFPE tissues, the morphology of frozen sections frequently is poor and further compromises the precision of microdissection. As mentioned above, IHC is an excellent tool for improving the visualization of the target populations. However, standard IHC protocols require several hours of incubation in aqueous media, which results in a significant loss of RNA through the action of ubiquitous RNases. Therefore, several groups have developed immunostaining protocols suitable for LCM and subsequent gene expression analysis. 17,25,26The following rapid immunostalning protocol can be performed with many different primary antibodies and standard reagents and renders mRNA of good quality, although a loss of mRNA in comparison to rapid routine staining (e.g., hematoxylin-eosin) does still occurY Immuno-LCM o f Frozen Sections. The Quick Staining kit (DAKO Corp., Carpinteria, CA) used in the initial publication is no longer commercially avallableY Alternatively, the procedure can be performed with analogous reagents suitable for quick immunostaining, such as a three-step (strept-)avidin-biotin 25E Fend,M. R. Emmert-Buck,R. Chuaqui,K. Cole,J. Lee,L. A. Liotta,andM. Raffeld,Am. J. Pathol. 154, 61 (1999). 26H. Murakami,L. Liotta,and R, A. Star, Kidney Int. 58, 1346(2000).
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FIG. 2. LCM of a frozen section of Hodgkin's disease immunostained for the CD30 antigen. The holes left behind are clearly visible, as well as the membrane staining of other tumor cells. The arrow denotes granulocytes, which show positivity due to endogenous peroxidase activity. (Rapid three-step immunoperoxidase technique, x 600.)
system with a biotinylated secondary antibody and horseradish peroxidase (HRP) (strept-)avidin complex optimized for high sensitivity (Fig. 2). A useful method is the employment of secondary antibodies coupled to a polymer backbone carrying multiple HRP molecules (EnVision, DAKO), which abolish the necessity for a third incubation step before color development and show increased sensitivity. Furthermore, the system is biotin-free and therefore lacks background staining as a result of endogenous biotin. 27
Staining procedure Frozen sections are mounted on charged slides (Superfrost Plus) and immediately refrozen on dry ice. Sections can be stored at - 8 0 °. The sections are briefly thawed at room temperature and immediately immersed in cold acetone for 1-2 min. Drying of the sections before fixation will severely compromise subsequent tissue capture. Do not stain more than 1-3 sections at one time, because multiple slides will lead to a significantly prolonged incubation time. After evaporation of acetone, the slides are rinsed briefly in buffered PBS or Tfis pH 7.4 and incubated with 70-100/zl primary antibody for 1.5-3 rain. 27 U. K'mmnerer, M. Kapp, A. M. Gassel, T. Richter, C. Tank, J. Dietl, and E Ruck, J. Histochem. Cytochem. 49, 623 (2001).
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The optimal dilution and staining time has to be determined individually. The slide is rinsed briefly with buffer and incubated with the secondary reagent for 2-3 min. If a three-step avidin-biotin system with a biotinylated secondary antibody is used, it is followed by incubation with the streptavidin-HRP complex for 2-3 min. Subsequently, the slide is covered with 100 /zl of freshly prepared 3,3'diaminobenzidine solution for 2-5 min. Then the slide is rinsed in water and briefly counterstained with hematoxylin (20-30 sec) if desired. The sections are dehydrated through graded alcohols (15-30 sec each, including twice 100% ethanol) and xylene (twice for 2 rain each). For all aqueous solutions, the use of pure, RNase treated water is recommended. During the incubation steps, placental RNase inhibitor (PerkinElmer) may be added in a concentration of 200-400 U/ml. RNA Extraction. After LCM, the tubes carying the caps with the captured cells are put on ice. RNA extraction is performed using lysis in guanidinium isothiocyanate followed by extraction with water-saturated phenol and subsequent precipitation with cold isopropanol and glycogen added as carrier (Micro RNA isolation kit, Stratagene, La Jolla, CA). If the presence of contaminating DNA is critical, DNase digestion is mandatory. RNA is dissolved in pure water containing 1 /zl of RNase inhibitor and incubated for 2 hr at 37 ° with 10-20 U of DNase I (Genhunter Corp., Nashville, TN). After reextraction of RNA following the same protocol as above, the RNA is dissolved in pure water, and 1/xl of RNase inhibitor is added. Reverse transcription is performed with 2.5/zmol/liter of random hexamers, 250/zmol/liter of each dNTP, and 100 U of MMLV reverse transcriptase (GenHunter) in a final volume of 20/zl. A mock reaction without RT should be performed in parallel.9,15 Depending on the amount of isolated cells, 20/zl of cDNA is usually sufficient for 10-20 or more single-step PCR reactions, and fragments larger than 500 bp can be amplified successfully. Conclusions The above protocols represent a small selection of fairly simple, easy-to-use techniques which can be readily performed in most molecular pathology laboratories. However, methodical improvements and further technical developments are realized at a rapid pace in all relevant areas, including tissue conservation and pre-LCM preparation, identification, and precise isolation of targeted cells, as well as more sensitive downstream analytical techniques adapted to small amounts of tissue, including high throughput screening methods such as cDNA microarrays or proteomics. Our increasing ability to correlate molecular findings with morphology and phenotype on the microscopic level will have a profound impact on all aspects of pathology.