Standard Protocol for Immunohistochemistry

Materials Required
1. Slides
2. Phosphate buffered saline (pH 7.6)
3. Hydrogen peroxide
4. Primary antibody
5. Blocking serum (normal serum)
6. Biotinylated secondary antibody
7. ABC reagent (6, 7, and 8 are included in Vectastain Elite ABC kit)
8. Diaminobenzidine
9. Meyer's hematoxylin
10.Permount

Method
1. Incubate in a dry oven at 62oC for 1 hour. Slides should be maintained in a vertical orientation
to allow complete removal of the paraffin.
2. Dewax slides in xylene for 5 x 4 minutes.
3. Hydrate slides in 100%, 95%, and 75% ethanol for 2 x 3 minutes each.
4. Immerse slides in tap water for 5 minutes
5. Antigen retrieval method (optional).
6. Quenching of endogenous peroxidase (optional)
a.immerse slides in 3% hydrogen peroxide solution for 6 minutes.
b.wash slides in PBS for 3 x 5 minutes.
7. Incubate slides with blocking serum (1:50) for 30 min.*
8. Blot excess serum from section, and incubate with primary Ab. Suggested incubation time
(may vary between antibodies):
mAb 2 hours at room temperature or overnight at 4oC.
pAb: 1 ~ 1.5 hours at room temperature.
9. Wash slides in PBS for 3 x 5 minutes.
10.Incubate slides with biotin-conjugated secondary Ab for 30 min.*
11.Wash slides in PBS for 3 x 5 minutes.
12.Incubate slides with Avidin-Biotin Complexes for 30 min.*
13.Wash slides in PBS for 3 x 5 minutes.
14.Incubate slides in fresh DAB solution for 2 minutes. (We use DAB solution in Vector
DAB/Ni substrate kit).**
15.Stop the reaction by washing in tap water.
16.Counterstain in Meyer's hematoxylin for 10 seconds.
17.Dehydrates slides in 75%, 80%, 95% and 100% ethanol
18.Clear slides in xylene 4 X 5 minutes.
19.Mount cover slide with Permount.


* Blocking serum, secondary antibody and avidin-biotin-peroxidase complexes are included in most of the
immunostaining kit. Our lab uses the ABC kit from Vector Lab (Vectastain Elite ABC kit).
** We use DAB solution in Vector/DAB/Ni substrate kit (Vector Labs., Cat. SK-4100).