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