J Clin Pathol 1983;36:903-906

Use of the cryostat section in electron microscopyP NORRIS, DWR GRIFFITHS

From the Department ofPathology, Weston Park Hospital, Whitham Road, Sheffield SI0 2SJ

SUMMARY A method using the cryostat section as a source of material from which relevant areasfor ultrastructural examination can be positively located and quickly processed is described.Preservation of the tissue does not appear to be unduly affected by freezing and subsequentcryostat sectioning.

The rapid processing technique as described byRowden and Lewis' could be considered a milestonein the processing of clinical material for electronmicroscopy. The method described by these authorsreduced the total processing time to three hoursinstead of the more conventional three to five days.2In the technique described below the processingtime is reduced again by half by using the cryostatsection, which unlike the standard electron micros-copy tissue block, permits precise localisation ofselected areas.The method retains the simplicity and precise

localisation of the paraffin wax section technique asdescribed by Gonzalez-Angulo et al, 3 together withthe preservation achieved by prompt glutaraldehydefixation.4Although relatively short processing techniques

are available5-7 this method will allow "late" speci-mens to be processed to Araldite resin within 30min, thus encouraging a more flexible and versatileelectron microscopy service.

Material and methods

Transverse slices of mouse kidney (3 mm thick)were frozen onto an OCT (manufactured by Labtekdivision of Miles laboratories) coated cryostat chuckusing a commercial dichlorodifluoromethane spray(Cryojet manufactured by Raymond A Lamb, Lon-don). Serial sections (10 ,m thick) were cut.

Light microscopic examination was performed onalternate sections after conventional air drying, for-malin fixation and haematoxylin and eosin stainingprocedures adopted for urgent biopsy work (Fig. 1).The remaining sections for electron microscopy

were fixed immediately, without air drying, inphosphate-buffered glutaraldehyde at 4°C for oneminute prior to processing. Specific localisationcould be obtained by examination of the unstained

Accepted for publication 30 March 1983

cryostat section following fixation using phase con-trast or interference microscopy.Areas of interest were located in the sections

stained for light microscopy and the correspondingareas relocated on the sections for electron micros-copy by scoring the underside of the slides. Areas ofinterest on sections examined by phase contrast mi-croscopy could be similarly identified. These sec-tions were then processed in the following manner:1 Rinse in 0-1 M sucrose phosphate

buffer 3 x 10s2 Osmicate in 2% unbuffered

osmium tetroxide 5 min3 Rinse in 99% alcohol 10 s4 Stain with saturated alcoholic

uranyl acetate 5 min5 Rinse in 99% alcohol 3 x 10 s6 Rinse in 100% alcohol 3 x 10 s7 Epoxy propane 6 x 10 s8 Araldite resin:epoxy

propane 50:50 2 x 3 min9 Araldite resin 5 min

Surplus resin was removed from around the edge ofthe selected area and the glass slide was theninverted (section downwards) on top of silicon rub-ber moulds (TAAB Enterprises, Reading) previ-ously filled with fresh Araldite resin. The resin wascured by polymerising at 100°C for one hour. Theblock with its attached slide was released from theembedding mould. The glass slide was removed byinserting a razor blade under the edge of the stillwarm block and detaching the block with theaccompanying section from the glass slide (Sokolet al8). The blocks were rapidly cooled by placinginto a -20°C deep freeze for a few minutes. Local-isation was re-established by examination of theblock face under a light microscope (Fig. 2), alterna-tively this could be achieved by cutting 1 gm sec-tions and staining with 1% toluidine blue. Afterappropriate orientation, 50 nm sections were thencut on a Reichert OM U2 ultramicrotome, floated

903

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Norris, Griffiths

Fig. 1 Haematoxylin and eosin stained cryostatsection featuring a single'mouse renalglomerulus. x 1100

.-

Fig. 2 The subsequent section in the sameserial group processed for electron microscopyand the glomerulus identified by viewing theblock surface before cutting ultrathinsections. x 1100

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Use of the cryostat section in electron microscopy

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Fig. 3 Low powered electronmaIcrograph ofa SO nm thicksection demonstrating the same

~<renal glomerulus. x 2000

Fig. 4 Electron micrograph ofanarea shown in Fig. 3. BM = basementmembrane; P = podocyte; FP = footprocesses (arrows) x 20000

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906out onto copper grids, stained with lead citrate9 andexamined with a Philips 400 T electron microscope(Fig. 3, 4).Results

See Figs. 1-4. On examination it appeared that thespecimens processed in the manner describedshowed no ultrastructural damage and ice crystalartefact was not observed. In view of the limitedamount of material available (10 ,um), orientationfor electron microscopy should be carried out byvisualising the block face rather than by cuttingsemi-thin sections for toluidine blue staining. Itshould be noted that when comparing the stainedcryostat section examined at light microscopy withthe corresponding area on the block face or thetoluidine blue stained section, the image is reversed.

Discussion

From the results obtained, it would seem that ultras-tructural damage is negligible when processingcryostat sectioned material for electron microscopystudies. By using the cryostat section as a source oftissue for electron microscopy, good localisation canbe achieved by comparing these sections with theserial sections cut and stained for light microscopy.

If phase contrast or interference microscopy hasbeen used on the glutaraldehyde fixed but unproces-sed cryostat section, specific localisation can beachieved equal to that attained by the removal ofselected areas from paraffin wax sections and betterthan that achieved when removing areas fromparaffin wax blocks, but with a degree of cellularpreservation similar to that of glutaraldehyde fixedtissue.Rapid processing can be adapted because of sec-

tion thickness, thus enabling electron microscopy tobe carried out within two hours of receipt.The cryostat section used in this way has consid-

erable potential. Areas worth exploring includeimmunocytochemistry and enzyme histochemistry,where standard methods could be performed on theglutaraldehyde-fixed sections before processingthrough to resin for electron microscopy. Labellingprocedures would require modification so thatsufficiently electron dense labels or final reactionproducts were used. With regard to enzyme his-tochemistry work has been carried out'0 localisinglysosomal and non-lysosomal acid phosphatase atelectron microscopical level by using the cryostatsection, but this was found inferior to equivalentwork using vibratome sections of nervous tissue.Further investigation is warranted in view of the farsuperior localisation afforded by the cryostat sec-tion.

Norris, GriffithsAnother instance where this method could prove

valuable is in the cytological study of fluid aspirates.By cutting cryostat sections of artificially-clottedfluid aspirates" routinely, a source of materialwould be available for electron microscopicalstudies if a situation occurred where light micros-copy gave an equivocal diagnosis of a poorly dif-ferentiated tumour. Although there are a number ofother techniques available to aid correlation ofcytological material between light and electron mic-roscopical levels-for example, split samples,stereoscopic examination of fixed or unfixed tissues,mirror imaging techniques-the technique describedhere guarantees that the tissue sample examined isexactly the same for light microscopy as it is forelectron microscopy.

In summary this technique may permit a numberof different histological investigations to be carriedout on a source of tissue where both light and elec-tron microscopical evaluation may be necessary.

The authors would like to thank the members ofstaff in the Pathology Laboratory at Weston ParkHospital for their help and critical assessment of themanuscript, Mrs JA Griffiths for her patience andtyping of the manuscript and Mrs J Rhodes for herphotographic expertise.

Referenm

' Rowden G, Lewis MG. Experience with a three hour electronmicroscopy service. J Clin Pathol 1974;27:505-7.

2 Luft JH. Improvements in epoxy resin embedding. J BiophysBiochem Cytol 1961;9:409-14.

3 Gonzalez-Angulo A, Ruiz de Chavez I, Castaneda M. A reliablemethod for electron microscopical examination. Am J ClinPathol 1978;70:697-9.

4 Sabatini DD, Bensch K, Barrnett RJ. Preservation of cellularultrastructure and enzyme activity and aldehyde fixation. JCell Biol 1963;17:19-58.

1 Robinson G. In: Bancroft JD, Stevens A, eds. Theory and prac-dce ofhistological technique. London: Churchill-Livingstone,1982:497.

6 Carr I, Toner PG. Rapid electron microscopy in oncology. J ClinPathol 1977;30:13-5.

7 Johannessen JV. Rapid processing of kidney biopsies for electronmicroscopy. Kidney Int 1973;3:46-50.

Sokol RJ, Norris PD, Hudson G. Examination of skdn windowpreparations by transmission electron microscopy. J ClinPathol 1980;33:697-8.

'Reynolds ES. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. l Cell Biol1963;17:208-12.

'° Wilks PN. An optimized lead capture electron histochemicaltechnique for the demonstration of lysosomal and non-lysosomal acid phosphatase activity in nervous tissue. MedLab Sci 1980;37:149-64.

Gregory PA. Cell block preparation: plasma-thrombin techni-que. Histo-Logic 1981;XI: 165.

Requests for reprints to: Mr P Norris, Department ofPathology, Weston Park Hospital, Sheffield S10 2SJ,England.

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