3 Micron Cryosections
- barthel
PROTOCOL FOR 3 MICRON CRYOSECTIONS
Barthel, L.K., and Raymond, P.A. (1990) Improved method for obtaining 3 micron cryosections for immunocytochemistry. J. Histochem. Cytochem. 38:1383-1388.
Works well for a range of 3-15 micron thick sections on both embryonic and adult tissue
Cryoprotection
Fix and rinse the tissue. After rinsing the tissue at room temperature in 0.1 M phosphate buffer with 5% sucrose, cryoprotect with increasing concentrations of sucrose by mixing 5% and 20% (0.6 M) sucrose on phosphate buffer in ratios of 2:1, 1:1, 1:2. Infiltrate the tissue for ½ hour in each sucrose mixture at room temperature, and then cryoprotect with 20% sucrose in phosphate at 4° C overnight. All fixation, rinses and infiltrations are done with gentle rotation.
Infiltration
Infiltrate the tissue in a mixture of 2 parts 20% sucrose phosphate buffer to 1 part O.C.T. embedding medium (Miles Inc.), for ½h at room temperature before freezing.
Embedding and Freezing
Transfer the tissue to an embedding mold fabricated from household aluminum foil, and fill the mold with fresh infiltration mixture (2:1, 20% sucrose:O.C.T.). Rapidly submerge the mold into isopentane cooled with liquid nitrogen. After the material is frozen wrap the block in cellophane and aluminum foil and store at -90° C.
Sectioning
3 μm sections are cut at -20° C. To achieve ideal sections it is critical that the knife edge be as sharp as possible. Trim the block face to a diamond shape, with the long axis oriented vertically. This orientation helps to make removal of the sections from the knife edge easier, and will minimize handling damage of the tissue. Use a small camel hair brush to guide the section off the block face and transfer it to a slide. Allow the section to dry on the slide at room temperature.
Slow the speed of sectioning (apprx. 5 mm/sec) to ease the manipulation of the sections as they are coming off the block face. Do not use the anti-roll plate furnished with the cryostat, it compresses the sections and results in poor tissue morphology.
Store the slides at -90°C until needed. Immediately prior to processing for immunocytochemistry remove the slides from the freezer, allow to warm to room temperature, and air dry. Do not freeze thaw slides. Only remove from the freezer what you plan to use.