Methods and Techniques Discussion => Immunohistochemistry (IHC) => Topic started by: ihcwor2 on March 12, 2003, 08:36:57 PM

Title: Indirect Immuno-POD Method (VIP) for Paraffin Sections
Post by: ihcwor2 on March 12, 2003, 08:36:57 PM
Indirect Immuno-POD Method (VIP) for Paraffin Sections

Solutions and Reagents

A.   10X Phosphate Buffered Saline (PBS):
To prepare 1 liter,
Na2HPO4 ------------------ 10.9 g
NaH2PO4 ------------------ 3.2 g
NaCl ------------------------ 90 g
Distilled water ----------- 1000 ml
Mix to dissolve and adjust pH to 7.4

B.   Antigen Retrieval Solution:
10mM Sodium Citrate Buffer (The most commonly used antigen retrieval reagent):
To prepare 1000 ml,
Sodium citrate ------------- 2.94 g
Distilled water ------------- 1000 ml
Adjust pH to 6.0

Selection of antigen retrieval techniques is crucial for successful staining of paraffin sections. Different antibodies may require different antigen retrieval techniques and need to be tested prior to application of actual projects.

C.   3% Hydrogen Peroxide:
To prepare 100 ml,
30% H2O2 ---------------- 10 ml
PBS or methanol ------- 90 ml

D.   Blocking Solution:
2% Normal Serum and 1% BSA in PBS:
To prepare 100 ml
Normal serum ---------- 2 ml
BSA ----------------------- 1 g
PBS ----------------------- 100 ml
Stir to dissolve.

E.   Primary Antibody:
Dilution of primary antibody is critical for success of staining, so a titration must be performed prior to application of actual projects.

F.   Secondary Antibody:
Peroxidase conjugated secondary antibody

G.   VIP Reagent


1.   Deparaffinize sections in xylene for 3x5min.
2.   Hydrate with 100% ethanol for 2x5min.
3.   Hydrate with 95% ethanol for 2x5min.
4.   Rinse in distilled water.

5.   Antigen Retrieval: (Select an appropriate antigen retrieval technique and it depends on antibodies used).
6.   Rinse sections in PBS for 1x5min.

7.   Hydrogen Peroxide: incubate sections in 3% H2O2 in PBS (or methanol) for 10-15 minutes to block endogenous peroxidase activity.
8.   Rinse in PBS for 1x2min.

9.   Blocking: incubate sections with 2% normal serum in PBS to block non-specific binding of secondary immunoglobulin.
10.   Rinse in PBS for 1x2min.                                                                        

11.   Primary antibody: incubate sections with primary antibody diluted in PBS for 1 hour at room temperature (A titer must be performed prior to actual projects)
12.   Rinse in PBS 3x5 min.

13.   Secondary antibody: incubate sections with peroxidase conjugated secondary antibody in PBS for 30 minutes at room temperature.
14.   Rinse in PBS for 3x5min.

15.   VIP: incubate sections with VIP solution for 2-10 minutes.
16.   Rinse in distilled water 2x5min.

17.   Counterstain with methyl green if desire.
18.   Rinse in distilled water 2x5min.
19.   Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x5min.
20.   Clear in xylene for 2x5min.

21.   Coverslip with mounting medium.


1.   Positive staining --------------- purple
2.   Nuclei -------------------------- green