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Techniques with resins other than glycol methacrylate
M~aronand Mic
aa~Laa~1cta, Vol. 16, No. 3, PP. 191—192, 1985.
Printed in Great Britain.
TECHNIQUES
WITH RESINS
OTHER
THAN GLYCOL
0739—6260/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd.
METHACRYLATE
Esther Roege Laboratory Michigan State University College of Veterinary Medicine E. Lansing, MI 48824 E.M.
ABSTRACT
At present, resin formulas other than the widely used Epon 812 mixtures are being successfully employed in electron microscopy laboratories at Michigan State University. The purpose of this presentation is to share information concerning materials and methods used in various electron microscopy laboratories at M.S.U. RESIN MIXTURES USED AT MSU The Electron Optic Center (EOC) uses a Spurrs—Mollenhauer #1 mixture, Dr. William Falls, Department of Anatomy, uses Maraglas 655 and the Veterinary Pathology Electron Microscopy Laboratory (VPEML) uses Araldite 502 (Richardson, 1960) SPURRS—MOLLENHAUER 4(1 After much experimentation in the EOC, the resultant Spurrs—Mollenhauer #1 (S—M#l) was found to combine the superior infiltration properties of Spurrs with the sectioning and staining properties of Mollenhauer #1. Batches are prepared biweekly, divided into single use quantities, and stored frozen in individual polypropylene beakers covered with parafilm. Large thick sections (1pm) and thin sections (800A°) of biological specimens of a wide range of characteristics can be produced using this embedding media. At present, Epon 812 is being used; therefore, when generic epon reagents are substituted the exact results will be unknown. MARAGLAS 655 Dr. William Falls uses a Maraglas 655 in a formula that results in an extremely hard block useful for 4 x 4mm blocks for thick sectioning, and trimmed to 1mm square for serial thin sectioning with glass knives. I have been unable to produce thick sections of 5 x 10 mm from blocks of this plastic. Because of the infiltration property, hardness and stainability, Maraglas 655 is a good product for nervous tissue. ARALDITE 502 Araldite 502, used in the VPEML, eliminates the use of the unavailable Epon 812, and results 0C maforwell block which Biological can be progressively thickinfiltrated and thin sectioning. specimens 2 hardened at 60—80 x 5 x 10 mm (Hill, Plopper, 1979), advantageous to the pathologist because of the larger size, are infiltrated with Araldite 502, embedded in molding cup trays and mounted on chucks compatible with the JB 4 microtome, which
191
192
E.
Roege
has been developed for large plastic block sectioning. After eight hours in a 65°C oven, the blocks are tested for hardness. One mm square areas of lesions to be studied are identified microscopically from thick sections stained with toluidine blue. The block is sawed into pieces containing the desired areas which can be held by chucks compatible with the ultramicrotome of choice. If the blocks are too soft for thin sectioning, they may be returned to the oven for further hardening. In some histochemical procedures requiring uniform results throughout the spec imen, 100 p m sections are cut with a vibratome, infiltrated, and embed— dad between a teflon coated slide and cover slip. Dr. Irene Crofova, Department of Anatomy, suggests the thinness of the gross specimen necessitates confinement on the teflon slide to ensure flattening. The specimen is polymerized in a 65°C oven, cut to 5 x 5 mm and glued to a blank polymerized plastic block for sectioning. Cytology samples may be concentrated by gentle centrifugation, a heated 1% noble agar and processed by routine methods.
embedded
in
In summary, Spurrs—Mollenhauer #1 is an excellent all purpose resin with good staining qualities and good infiltration, Maraglas 655 ptoduces a hard block useful for serial sectioning nervous tissue and Araldite 502 polymerizes quickly and large thick sections can be prepared before thin sectioning. ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. Tracie Bun ton encouragement and help as a mentor of high standards. Dr. Karen of the EOC, Dr. William Falls and Dr. Irene Grofova, Department of and Stuart Pankratz, Department of Microbiology were very generous the
techniques
developed
in
their
for her K. Baker Anatomy to share
laboratories.
REFERENCES Hill, L.H., and C.G. Plopper (1979). Use of large block embedding for correlated LM, TEM, and SEM characterization of pulmonary airways of known anatomic
location.
Richardson, resins
313—323.
for
Proc.
K.C., L. ultratliin
Southeast
Elec.
Microsc.
Soc.,
2,
24.
Jarett, and E.H. Finke (1960). Embedding in epoxy sectioning in electron microscopy. Stain Tech., 35,
aa~Laa~1cta, Vol. 16, No. 3, PP. 191—192, 1985.
Printed in Great Britain.
TECHNIQUES
WITH RESINS
OTHER
THAN GLYCOL
0739—6260/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd.
METHACRYLATE
Esther Roege Laboratory Michigan State University College of Veterinary Medicine E. Lansing, MI 48824 E.M.
ABSTRACT
At present, resin formulas other than the widely used Epon 812 mixtures are being successfully employed in electron microscopy laboratories at Michigan State University. The purpose of this presentation is to share information concerning materials and methods used in various electron microscopy laboratories at M.S.U. RESIN MIXTURES USED AT MSU The Electron Optic Center (EOC) uses a Spurrs—Mollenhauer #1 mixture, Dr. William Falls, Department of Anatomy, uses Maraglas 655 and the Veterinary Pathology Electron Microscopy Laboratory (VPEML) uses Araldite 502 (Richardson, 1960) SPURRS—MOLLENHAUER 4(1 After much experimentation in the EOC, the resultant Spurrs—Mollenhauer #1 (S—M#l) was found to combine the superior infiltration properties of Spurrs with the sectioning and staining properties of Mollenhauer #1. Batches are prepared biweekly, divided into single use quantities, and stored frozen in individual polypropylene beakers covered with parafilm. Large thick sections (1pm) and thin sections (800A°) of biological specimens of a wide range of characteristics can be produced using this embedding media. At present, Epon 812 is being used; therefore, when generic epon reagents are substituted the exact results will be unknown. MARAGLAS 655 Dr. William Falls uses a Maraglas 655 in a formula that results in an extremely hard block useful for 4 x 4mm blocks for thick sectioning, and trimmed to 1mm square for serial thin sectioning with glass knives. I have been unable to produce thick sections of 5 x 10 mm from blocks of this plastic. Because of the infiltration property, hardness and stainability, Maraglas 655 is a good product for nervous tissue. ARALDITE 502 Araldite 502, used in the VPEML, eliminates the use of the unavailable Epon 812, and results 0C maforwell block which Biological can be progressively thickinfiltrated and thin sectioning. specimens 2 hardened at 60—80 x 5 x 10 mm (Hill, Plopper, 1979), advantageous to the pathologist because of the larger size, are infiltrated with Araldite 502, embedded in molding cup trays and mounted on chucks compatible with the JB 4 microtome, which
191
192
E.
Roege
has been developed for large plastic block sectioning. After eight hours in a 65°C oven, the blocks are tested for hardness. One mm square areas of lesions to be studied are identified microscopically from thick sections stained with toluidine blue. The block is sawed into pieces containing the desired areas which can be held by chucks compatible with the ultramicrotome of choice. If the blocks are too soft for thin sectioning, they may be returned to the oven for further hardening. In some histochemical procedures requiring uniform results throughout the spec imen, 100 p m sections are cut with a vibratome, infiltrated, and embed— dad between a teflon coated slide and cover slip. Dr. Irene Crofova, Department of Anatomy, suggests the thinness of the gross specimen necessitates confinement on the teflon slide to ensure flattening. The specimen is polymerized in a 65°C oven, cut to 5 x 5 mm and glued to a blank polymerized plastic block for sectioning. Cytology samples may be concentrated by gentle centrifugation, a heated 1% noble agar and processed by routine methods.
embedded
in
In summary, Spurrs—Mollenhauer #1 is an excellent all purpose resin with good staining qualities and good infiltration, Maraglas 655 ptoduces a hard block useful for serial sectioning nervous tissue and Araldite 502 polymerizes quickly and large thick sections can be prepared before thin sectioning. ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. Tracie Bun ton encouragement and help as a mentor of high standards. Dr. Karen of the EOC, Dr. William Falls and Dr. Irene Grofova, Department of and Stuart Pankratz, Department of Microbiology were very generous the
techniques
developed
in
their
for her K. Baker Anatomy to share
laboratories.
REFERENCES Hill, L.H., and C.G. Plopper (1979). Use of large block embedding for correlated LM, TEM, and SEM characterization of pulmonary airways of known anatomic
location.
Richardson, resins
313—323.
for
Proc.
K.C., L. ultratliin
Southeast
Elec.
Microsc.
Soc.,
2,
24.
Jarett, and E.H. Finke (1960). Embedding in epoxy sectioning in electron microscopy. Stain Tech., 35,