A Highly Sensitive IHC Detection Protocol (HRP/DAB)

Polymer Enhanced Method

A. Jamsa from Carolinska University

 

GBI Polink Polymer Detection Systems

 

 

 

Introduction:

 

This protocol is good for detecting both mouse and rabbit primary antibodies on human tissues. It is very sensitive and stable.

 

Reagents:

Solution A (antibody linker)
Prepared properly, the solution will be stable for 18 months at 4 degrees. 
  • Rabbit anti-Mouse IgG (min x human serum protein) (Jackson ImmunoResearch Cat No. 315-005-045). Working concentration: 1-2ug/ml
  • Normal Goat Serum (Jackson ImmunoResearch Cat No. 005-000-121). Working concentration: 1%
  • Working Solution: Add concentrated rabbit anti-mouse IgG, normal goat serum, in antibody diluent (1% BSA, 0.1% sodium azide, in pH7.4 PBS) and make into working solution.
Solution B (polymer HRP anti-Rabbit)
You may buy this solution from many vendors: DAKO envision, Vector ImmPress etc.
 
Procedures:
After primary antibody incubation, apply Solution A on tissue slides and incubate 20 minutes under room temperature. Then wash in PBS wash buffer 3 minutes x 3. Apply Solution B and incubate for 30 minutes under RT, wash in PBS wash buffer 3 minutes x 3. Apply chromogen, and following other routine procedures.
 
Notes: This detection protocol is very sensitive for both mouse and rabbit primary antibodies. It is a good protocol for labs with routine IHC.

 

References: