Y10B IMMUNOCYTOCHEMISTRY on Paraffin

Fixation.

- Y10b will stain tissue fixed in Bouin's, Methacarn or 4% paraformaldehyde.

- Sections can be cut on vibratome, cryostat or paraffin microtome. Plastic

embedded tissue has not been tested.

-Tissue culture cells are best stained by fixing with 4% para. and permeabilizing with 0.1% saponin

Mounting.

Mount paraffin sections on acid washed chrome alum subbed slides and dry overnight at 40° C.

Deparaffinize.

1. Xylene 10 min. X3

2. 100% EtOH, 3 min. X2

3. 95% EtOH, 3 min.

4. 70% EtOH, 3 min.

5. PBS, 10 min. to 1 hour.

Immunocytochemistry.

1. Block with 4% normal horse serum in PBS/0.5% BSA for 1 hour;

2. Primary antibody is usually in a dilution range from 1:10 to 1:500, leave on overnight;

3. Wash in PBS, 10 minutes X3;

4. Secondary antibody:1:400 biotintylated horse anti-mouse IgG;

5. Wash in PBS, 10 min. X3;

6. Make ABC solution 30 minutes prior to adding:

use 10ml PBS without azide, add 2 drops of solution A, mix, add two drops of solution B and mix.

7. 1 hour incubation with ABC solution;

8. Wash in PBS, 10 min X3;

9. DAB reaction: Add 1 ml of 2.25 mg/ml DAB solution to 5 ml of Tris-HCl buffer, pH 7.6;

Just before adding to slides, add 5ml of H2O2;

10. Incubate 10 min. with DAB;

11. Wash with PBS, 10 min X3

12. Dehydrate and coverslip as usual.

Reference : Learner et. al., (1981) P.N.A.S. 78:2737-2741.

Gwenn Garden, Karen Canady and Ed Rubel, 1991