I am doing IHC using an ABC staining kit (centacruz), concentration of primary and secondary antibody is optimized to detect the desired protein, incubate DAB 30 min, counterstain 5 second, but the background in the slide is also stained which I cannot remove with more washing.
Dear Mehr
Since you are using ABC staining kit you must known that some cells or tissues, such as kidney, liver, spleen, intestine, colon, and prostate, contain endogenous biotin. Using Avidin-Biotin detection methods may results in non-specific background staining. This non-specific background can be significantly reduced by pre-treatment of cells or tissues with Avidin/Biotin Blocking Solution prior to the incubation of biotinylated antibody. Also, you need to block the non specific proteins and the endogenous peroxidase that give false results (stained background).
My recommendations are:
1.Prepare fresh hydrogen peroxide for blocking endogenous peroxidase
2.Use a protein blocking serum same as secondary antibody or you can use Bovine serum albumin
3. Increase the incubation time of avidin/biotin blocking solution
4. As mohamed said, reduce the time of DAB and you can select the desired color by checking sections under the microscope.
5. Finally, check the antibody dilution titre.
6. Also, You can use positive and negative slides
Best regards
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining