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Observations on the osmium tetroxide-p-phenylenediamine staining method for Epon semithin sections of nervous tissue
Ada hiKtudlf'm. 72, 123-125 (19H3)
Dppa!"tanwnto dt' Cito!og[,t" Histologia, Fa('ultad (]t' Cit'Il('ias, FnivArsidad Autonoma de Madrid, Hpain
Short communication
Observations on the osmium tetroxidep-phenylenediamine staining method for Epon semithin sections of nervous tissue By
ONDINA COLMAN
and
.JUAN C. fhoCKERT
Introduction An increasing variC'ty of general, selective. and cytochemical staining techniques is now available for plastic-embedded semithin sections (for references see CANETE and STOCKERT 1981). Although somc of these t~chniques give a good differentiation of components in nervous tissue under the light microscope. preciRc correlations at the ultrastructural level are difficult to achieve. On the other hand. the advantages of processing simultaneously samples of ncrvous tiRRues for light and electron microscopic observations are obvious. 0140 4 is a well known contrasting agent for lipids (HAY AT 1975) and RN A containing structures (STOCKERT and COLMAN 1974; STOCKERT 1977). After 0140 4 fixation a better visualization of cell component~ is obtained by treatment with p-phenylenediamine (PPD) (ESTA'BLE-PUW et al. 1965; LEDINGHAM and SIMPSOX 1972; STOCKERT 1977; ESPONDA and STOCKERT 1978). The aim of this communication is to comm~nt the staining m~chaniRm and to emphasize the potential of the OS04-PPD procedure as a simple and useful technique in neurohistological re8earch.
Material and methods nufo sp. we!"t' fixt'd in (\.5 % glutar'aldehyde
Hamples of spinal ('01'<1 f!'Om in SORENSEN'S buffer at pH = (\.S for 3 hand pm;tiixpd III 2 ~;) 0040" in 1i.5 (Yo SlH'fOSe solution for 3 h. After ethanol dt'hydration, the Kalllples wt'rt' t'mbt'ddp([ in Epoll as usual. Remithin Redions (1 to 211m) were tl'ansfprr'ed to " dmp of water on a slide and then dripd on it hot plate. Preparations were treated with H freshly madt' 0.5 % aqueous p-plwnylenedialllinp-(l'PD)-(.Merrk) solution at (\O°C for 30 to (\0 min. After wHshing in distill"d wat('r and air-drying they werA mountpd in Sandeuml or Euk·itt and obs('t'vP(i Ilndpt' bright fipld illumination.
Results and Discussion After thi:-; :-;imple procedure, fine structural details in nerVOUfl tissue are differentiated in an excellent way. The NrssL material stains greyi~h brown, nuclei take up a brown colour, and nucleoli appear as black bodies. The cytoplaRm of glial cells stains light 9*
124
0. COLMAN and .J. C. STOCKERT
ochre, while nuclei appear violet. The neuropil area shows a pink to pale violet shade and the myelin sheaths stain dark violet to black. A finely granular texture of the chromatin is apparent in neurons; on the contrary, several masses of condensed chromatin can often be found in glial cell nuclei. On the luminal surface of the neural canal, cilia from ependymal cells are also observed as dark threadR. ThiR method was applied to examine the histology of the spinal cord but perypheral nerves and other portions of the central nervous system could also be successfully studied, showing the same staining characteristics as described. Thus, a suitable visualization of cytological details, and possibilities to correlate light and electron microscopic observations make the glutaraldehyde-Os0 4-PPD procedure a useful general staining technique for Epon semithin sections. Rt'lated to the interprt'tation of this staining pattern is the well known contrasting propt'rties of OS04 for lipids (t'.g. myelin sheaths). Howevt'r, Os04 also rt'acts with the doublt' bond of pyrimi· dines in singlt'.stranded nuclt'ic acids, forming the addition product 5,6·cis-diol oRmium (VI) {)xidt' (BURTON and RILEY 1966)_ This reaction has been used in eledron microscopy (WHITING and OTTENSMEYER 1972), and in crystallographic analysis of transfer RNA (RosA and SIGLER 1974). At the cytological level, the reaction of Os04 with RNA (STOCKERT 1977) could also account for the osmiophilia of nuC'leoli, basophilie cytoplasm and other RNA containing structurt'R (\VIGGLESWORTH 1957; BATTAGLIA and MAGGINI 1968; STOCKERT and COLMAN 1974; ESPONDA and STOCKERT 1978; COLMAN and STOCKERT 1979)_ Little is known about how a previous aldehyde fixation modifieH the osmiophilia of ct'll structures. Glutaraldehyde introduces free aldehyde groups in tissues (KASTEN 1965), which later react with OS04 (HOPWOOD 1970); this mechanism could explain the intense staining reaction in chromatin. Some interactions of OS04 with proteins, involving -SH groups (HAKE 19(5) and aromatic bonds in tryptophane and histidine (PORTER and KALLMAN 1953; RUTHMANN 1970), are also pxpected to contributt' to tht' observed staining pattern. On thp other hand, PPD product'S a striking blackening in OS04 fixed tissues because of the rt'action with bound but unrt'duced osmium (LEDINGHAM and SIMPSON 1972). Semithin st'ctions from glutaraldehyde-Os0 4 fixed tissues only show a pale yellow colour, which becomes dark ochrebrown after treatment with PPD_ In addition to the reduction of bound osmium to black oxides, an increased Rtaining degree could further originate from the coupled deposition of dark-coloured oxidation products of PPD (LISON 1960; GABE 19(8), and from their dirt'ct interaction with polyanionic Rubstrates (STOCKERT et ai. 1980).
Acknowledgements This work was supported by a grant (No_ 4142-79) from the Comision Asesora de lnvestigacion 'Cientifica y Tecnica, Spain_ The authors are indt'bted to Mrs. R. TORRENT for her valuable collaboration.
Literature BATTAGLIA, E., and MAGGINI, F_, Tht' URt' of osmium for staining the nucleolus in squash tt'chnique_ Caryologia 21, 287 -292 (19G8)_ BURTON, K., and RILEY, \V. T., Selective dt'gradatation of thymidine and thymidine deoxynuclt'otides. Biochem_ J_ 98, 70-77 (1966). CANETE, M., and STOCKERT, .T. C., Polychromatic GIEMSA staining for Epon semi-thin sections_ J_ Microscopy 122, 321-325 (1981).
Observations on the osmiulll tetroxide
125
('OL:Il.\N, O. D., and STOCKERT, .J. C., Elt'etron llli('],OH('OPY of synaptonemal ('omplf'xf's in sf'lllithin N(,(,tionH. Z, Naturfor::wh. 34e, 299-300 (1979). EHPOND,\, P., and STOCKERT, .J. C., Localization of the synaptonf'mal ('omplf'x ullwooJ), n., The readionH betwe"l1 fOI'maldphyde, glutaraldehyde and osmium tetroxide, and their fixation effpt'is on bovinp senllll albumin and on tis";lH' b10eks. Histoehemie 24, 50-64 (1970). K \NTEN, F. H., l'l'efprential bin(ling of glutamldehy(le and acrolein to nuclei .•1. Histo('ltelll. Cyto('helll. 13,30-31 (HHi5). LEDINGHAM, .J. M., and Sn1PsoN, F. 0., Thp use of p·phenylelwdiamine in the blQ('k to enhance osmium Ntaining for e1eetron mief'o";('oPY. Rtain Technol. 47, 239-·243 (1972). LISON, L., Histo('llPllli(' et ('ytoellPmi(> ,mimalps, Principes et methods. Vol. 1, Gauthipr-Villars, Paris 19(iO. POWl'ER, K. R" and KALLlVL\N, F., l'roppl'tips and "ffpeis of osmium tptroxide as a tissue fixative with ,.;pe('ial l'eferE'nl'e to its use for d(~('tron lIli!'ros('opy. Exptl. Cell Rps. 4, 127 -141 (1953). HONA, .J •• J., ami SIGLER, P. B., Thp sit!' of ('ovalE'nt attaf'hnwnt in tIlE' osmium -tRNA r met ISOmOl'ph011N (kt'ivatiw. Bi(wllemistl'Y 13, 5102- 5110 (1974). R'l'OCKERl', ,J. C., ONllIium tf'tl'oxi
Dppa!"tanwnto dt' Cito!og[,t" Histologia, Fa('ultad (]t' Cit'Il('ias, FnivArsidad Autonoma de Madrid, Hpain
Short communication
Observations on the osmium tetroxidep-phenylenediamine staining method for Epon semithin sections of nervous tissue By
ONDINA COLMAN
and
.JUAN C. fhoCKERT
Introduction An increasing variC'ty of general, selective. and cytochemical staining techniques is now available for plastic-embedded semithin sections (for references see CANETE and STOCKERT 1981). Although somc of these t~chniques give a good differentiation of components in nervous tissue under the light microscope. preciRc correlations at the ultrastructural level are difficult to achieve. On the other hand. the advantages of processing simultaneously samples of ncrvous tiRRues for light and electron microscopic observations are obvious. 0140 4 is a well known contrasting agent for lipids (HAY AT 1975) and RN A containing structures (STOCKERT and COLMAN 1974; STOCKERT 1977). After 0140 4 fixation a better visualization of cell component~ is obtained by treatment with p-phenylenediamine (PPD) (ESTA'BLE-PUW et al. 1965; LEDINGHAM and SIMPSOX 1972; STOCKERT 1977; ESPONDA and STOCKERT 1978). The aim of this communication is to comm~nt the staining m~chaniRm and to emphasize the potential of the OS04-PPD procedure as a simple and useful technique in neurohistological re8earch.
Material and methods nufo sp. we!"t' fixt'd in (\.5 % glutar'aldehyde
Hamples of spinal ('01'<1 f!'Om in SORENSEN'S buffer at pH = (\.S for 3 hand pm;tiixpd III 2 ~;) 0040" in 1i.5 (Yo SlH'fOSe solution for 3 h. After ethanol dt'hydration, the Kalllples wt'rt' t'mbt'ddp([ in Epoll as usual. Remithin Redions (1 to 211m) were tl'ansfprr'ed to " dmp of water on a slide and then dripd on it hot plate. Preparations were treated with H freshly madt' 0.5 % aqueous p-plwnylenedialllinp-(l'PD)-(.Merrk) solution at (\O°C for 30 to (\0 min. After wHshing in distill"d wat('r and air-drying they werA mountpd in Sandeuml or Euk·itt and obs('t'vP(i Ilndpt' bright fipld illumination.
Results and Discussion After thi:-; :-;imple procedure, fine structural details in nerVOUfl tissue are differentiated in an excellent way. The NrssL material stains greyi~h brown, nuclei take up a brown colour, and nucleoli appear as black bodies. The cytoplaRm of glial cells stains light 9*
124
0. COLMAN and .J. C. STOCKERT
ochre, while nuclei appear violet. The neuropil area shows a pink to pale violet shade and the myelin sheaths stain dark violet to black. A finely granular texture of the chromatin is apparent in neurons; on the contrary, several masses of condensed chromatin can often be found in glial cell nuclei. On the luminal surface of the neural canal, cilia from ependymal cells are also observed as dark threadR. ThiR method was applied to examine the histology of the spinal cord but perypheral nerves and other portions of the central nervous system could also be successfully studied, showing the same staining characteristics as described. Thus, a suitable visualization of cytological details, and possibilities to correlate light and electron microscopic observations make the glutaraldehyde-Os0 4-PPD procedure a useful general staining technique for Epon semithin sections. Rt'lated to the interprt'tation of this staining pattern is the well known contrasting propt'rties of OS04 for lipids (t'.g. myelin sheaths). Howevt'r, Os04 also rt'acts with the doublt' bond of pyrimi· dines in singlt'.stranded nuclt'ic acids, forming the addition product 5,6·cis-diol oRmium (VI) {)xidt' (BURTON and RILEY 1966)_ This reaction has been used in eledron microscopy (WHITING and OTTENSMEYER 1972), and in crystallographic analysis of transfer RNA (RosA and SIGLER 1974). At the cytological level, the reaction of Os04 with RNA (STOCKERT 1977) could also account for the osmiophilia of nuC'leoli, basophilie cytoplasm and other RNA containing structurt'R (\VIGGLESWORTH 1957; BATTAGLIA and MAGGINI 1968; STOCKERT and COLMAN 1974; ESPONDA and STOCKERT 1978; COLMAN and STOCKERT 1979)_ Little is known about how a previous aldehyde fixation modifieH the osmiophilia of ct'll structures. Glutaraldehyde introduces free aldehyde groups in tissues (KASTEN 1965), which later react with OS04 (HOPWOOD 1970); this mechanism could explain the intense staining reaction in chromatin. Some interactions of OS04 with proteins, involving -SH groups (HAKE 19(5) and aromatic bonds in tryptophane and histidine (PORTER and KALLMAN 1953; RUTHMANN 1970), are also pxpected to contributt' to tht' observed staining pattern. On thp other hand, PPD product'S a striking blackening in OS04 fixed tissues because of the rt'action with bound but unrt'duced osmium (LEDINGHAM and SIMPSON 1972). Semithin st'ctions from glutaraldehyde-Os0 4 fixed tissues only show a pale yellow colour, which becomes dark ochrebrown after treatment with PPD_ In addition to the reduction of bound osmium to black oxides, an increased Rtaining degree could further originate from the coupled deposition of dark-coloured oxidation products of PPD (LISON 1960; GABE 19(8), and from their dirt'ct interaction with polyanionic Rubstrates (STOCKERT et ai. 1980).
Acknowledgements This work was supported by a grant (No_ 4142-79) from the Comision Asesora de lnvestigacion 'Cientifica y Tecnica, Spain_ The authors are indt'bted to Mrs. R. TORRENT for her valuable collaboration.
Literature BATTAGLIA, E., and MAGGINI, F_, Tht' URt' of osmium for staining the nucleolus in squash tt'chnique_ Caryologia 21, 287 -292 (19G8)_ BURTON, K., and RILEY, \V. T., Selective dt'gradatation of thymidine and thymidine deoxynuclt'otides. Biochem_ J_ 98, 70-77 (1966). CANETE, M., and STOCKERT, .T. C., Polychromatic GIEMSA staining for Epon semi-thin sections_ J_ Microscopy 122, 321-325 (1981).
Observations on the osmiulll tetroxide
125
('OL:Il.\N, O. D., and STOCKERT, .J. C., Elt'etron llli('],OH('OPY of synaptonemal ('omplf'xf's in sf'lllithin N(,(,tionH. Z, Naturfor::wh. 34e, 299-300 (1979). EHPOND,\, P., and STOCKERT, .J. C., Localization of the synaptonf'mal ('omplf'x ull