Methods and Techniques for Frozen Tissue Sections
Procedure
-
Snap frozen fresh tissues in liquid
nitrogen or isopentane pre-cooled in liquid nitrogen, embedded
in OCT compound in cryomolds. Store frozen blocks at - 80 ºC.
Alternately one can use
precision cryoembedding system to perform
fresh tissue embedding.
-
Cut 4-8 um thick cryostat sections and
mount on superfrost plus slides or gelatin coated slides. Store
slides at - 80 ºC until needed. The slides can be store at -20
ºC for short term storage (within a few weeks).
-
Before staining, warm slides at room
temperature for 30-60 minutes and fix in ice cold acetone or
other alternate fixatives for 5-10
minutes. Air dry for 30-60 minutes.
-
Wash in PBS or TBS and proceed to standard staining procedure.
Principal Factors for Good Sectioning of Frozen
Specimens
-
The temperature must be correct for the specimen being cut.
-
The microtome must be correctly adjusted
and operated.
-
The cutting blade must be sharp and set
at the correct angle.
-
The anti-roll plate must be correctly adjusted.
Recommended Temperatures for Cutting Unfixed Frozen
Tissues
| Tissue Type |
Working
Temperature |
| Brain |
-12 ºC |
| Liver |
-14 ºC |
| Lymph Node |
-14 ºC |
| Kidney |
-16 ºC |
| Spleen |
-16 ºC |
| Muscle |
-20 ºC |
| Thyroid |
-20 ºC |
| Skin |
-25 ºC |
| Breast |
-25 ºC |
| Breast with Fat |
-30
ºC or below |
| Adipose Tissue |
-30
ºC or below |
| Fixed Tissue |
-12 ºC
to -17 ºC |
