Staining Procedures For Plastic Embedded Tissue
Verified at the Applications Laboratory of the Biomedical Division,
Sorvall Microtomes
SOLUTIONS:
| Solution A: Sodium Chlorite
Solution |
|
2.19g |
|
Sodium chlorite |
|
78.0ml |
|
Distilled water |
|
|
|
|
| Solution B: 5 N Acetic Acid |
|
28.57ml |
|
Glacial acetic acid |
|
100.0ml |
|
Distilled water |
|
|
|
|
| Blockade Working Solution |
|
All |
|
Solution A |
|
20.0ml |
|
Solution B |
BLOCKING PROCEDURE:
- Treat slides from overnight to 48 hours.
- Rinse well in tap water.
- Proceed to Schiff stains.
b. Feulgen Reaction for Nucleic Acids (Refs 5,3)
SOLUTIONS:
STAINING PROCEDURE:
- Perform aldehyde blockade in chlorous acid.
- Wash in running tap water for 10 minutes.
- Rinse in distilled water for 2 minutes.
- Treat in HCl at 60ºC for 10 minutes.
- Rinse in distilled water for 5 minutes.
- Stain in Schiff reagent for 15-20 minutes.
- Wash in running tap water for 20 minutes.
- Rinse in distilled water for 2 minutes.
- Counterstain in fast green at room temperature
for ten seconds for yellow stain; or at 60ºC for 10 seconds for
green stain.
Rinse in distilled water to clear plastic for 5 seconds.
Blow dry.
Mount
RESULTS:
Nuclear chromatin and some inclusion bodies
-- red; other structures -- yellow or green depending on the method
of counterstaining.
SOLUTIONS:
| Aldehyde Blockade with Chlorous
Acid |
|
|
|
See formula in procedure 2-10,
a. |
|
|
|
|
| 1% Periodic Acid |
|
1.0g |
|
Periodic acid |
|
100.0ml |
|
Distilled water |
|
|
|
|
| Schiff Reagent |
|
|
|
Use commercial solution or see formula
in procedure 2-10, d |
|
|
|
|
| Sulfurous Acid |
|
5.0ml |
|
Hydrochloric acid, 1 N |
|
5.0ml |
|
Sodium metabisulfite, 10% |
|
100.0ml |
|
Distilled water
Make fresh. |
|
|
|
|
| 0.3% Methyl Green |
|
0.15g |
|
Methyl green |
|
50.0ml |
|
Ethyl alcohol, 95% |
|
|
|
|
| 0.5% Acetic Acid |
|
0.5ml |
|
Glacial acetic acid |
|
100.0ml |
|
Distilled water |
|
|
|
|
| Harris' Hematoxylin |
|
|
|
See formula in procedure 2-7,
b |
|
|
|
|
STAINING PROCEDURE:
- Perform aldehyde blockade in chlorous acid.
- Wash in running tap water for 10 minutes.
- Rinse in distilled water for 2 minutes.
- Treat in periodic acid for 10 minutes.
- Wash in distilled water for 5 minutes.
- Stain in Schiff reagent for 15-20 minutes.
- Bleach in sulfurous acid for 2 minutes.
- Repeat.
- Rinse in distilled water for 5 minutes.
Counterstain in methyl green for 3-7 minutes.
Destain in acetic acid.
Rinse in distilled water.
Blow dry.
Mount.
OR:
Counterstain in Harris' hematoxylin for 10 minutes.
Follow routine procedure to distilled water (see
procedure 2-7, b, omitting steps 8 and 9).
Blow dry.
Mount.
RESULTS:
Polysaccharides -- red; other tissue components stained according
to the counterstain.
|
900.0ml |
|
Distilled water
Boil in 2 liter flask. Slowly add: |
|
5.0g |
|
Basic fuchsin
Swirl for 1 minute. Vacuum filter through two layers of filter
paper in a buchner funnel into a 1 liter flask. Cool to 50ºC.
Slowly, while swirling, add: |
|
100.0ml |
|
Hydrochloric acid, 1.0 N |
|
10.0g |
|
Potassium metabisulfite, K2S2O5
Swirl 2 minutes. Cloudy red solution will become clear blood red. Stopper
and store in refrigerator for 24 hours. Then remove from refrigerator,
warm to room temperature, and add: |
|
3.75g |
|
Activated charcoal
Shake vigorously for 1 minute. Vacuum filter immediately. The reagent should
be clear and colorless. If not, repeat the charcoal step. Store Schiff
reagent in the refrigerator. May be used in 48 hours; remains stable for
months. |
Warning: Some of the chemicals
used for the staining procedures given in this section may be hazardous
if misused. For this reason, read and observe all warnings and cautions
provided by the manufacturer for each chemical before proceeding
with a staining procedure.
Note: In order to prevent sections from loosening
from the slides during staining, all sections should be heat-fixed
(60ºC to 100ºC)
to the slides for a minimum of 2-5 minutes prior to staining, preferably
at the time the sections are mounted on the slides.
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