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| 1. |
Dissect tissue no larger than 1 cm2 x 0.4 cm. |
| 2. |
Put the specimen on the fringe of a card strip and label the card. Put it in liquid nitrogen for 60 seconds and bring it out to put on dry ice. Cut the card
so that it is larger size than that of the specimen and transfer to a freezing vial cooled to -70℃ in advance. |
| 3. |
After sectioning, coat a glass slide with 1% gelatin. Heat gelatin in 50℃ water and cool down. Add 0.02% sodium azide. Soak slides in gelatin solution for 30
seconds and air-dry. |
| 4. |
Make frozen tissue section using cryostate. Each section should be 5 ㎛ to 10 ㎛ thick. Collect sections on coated slides. |
| 5. |
Air-dry sections and then soak in fresh 4% paraformaldehyde for 2 minutes. |
| 6. |
Wash out with PBS several times and then soak in 1% NP-40 for 5minutes. |
| 7. |
Wash out with PBS several times. |
| 8. |
Place a slide with tissue section in a humidified chamber and soak for blocking in 3 % normal serum in PBS for 30 minutes. |
| 9. |
After removing serum, add first antibody solution without washing. Make sure to use protein-containing solutions like 3% BSA in PBS in dilution. The dilution
fold should follow the manual of products. Without knowing the dilution fold, test the serial dilution of 1/10, 1/100, 1/1000, 1/10000. |
| 10. |
Incubate the slide for at least 60 minutes at room temperature in a humidified chamber. To increase sensitivity, the incubation time may be extended to 24 hours. |
| 11. |
Wash out with PBS three times for 5 minutes each. |
| 12. |
Prepare a horseradish peroxidase-labeled secondary antibody against the primary antibody. |
| 13. |
Incubate the secondary antibody in humidified chamber for 30 minutes at room temperature. |
| 14. |
Wash out with PBS three times for 5 minutes each. |
| 15. |
Prepare DAB/metal reagent. Dissolve 6mg of DAB in 9ml of 0.05M Tris buffer(pH 7.6), and then add 1ml of 0.3% wt/vol stock solution of nickel chloride in water
and 0.1ml of 3% hydrogen peroxide in water. |
| 16. |
Treat specimen with the reagent. Inspect the sample under low-power light microscope. If enough precipitation reaction of brown/black happen, stop the reaction
with water. It takes 1 to 20 minutes in general. |
| 17. |
Add some drops of Harris?hematoxylin to the specimen, incubate for 5 minutes. |
| 18. |
Wash the slide gently with water. |
| 19. |
Dehydrate the sample by passing through graded alcohol. React with each alcohol twice for 3 minutes each and air-dry in the order of 75% ethanol, 95% ethanol
and absolute ethanol. |
| 20. |
Drop a small amount of DPX on the specimen and place the cover slip carefully not to make air-bubble. Remove surplus mounting medium with paper towel and inspect. |
