I have some tissue cultures cells and I want to perform IHC for these cells using DAB as a chromogenic solution. I want to ask if the peroxidase blocking is necessary in this staining or not?
Hi Mohamed,
For immuohistochemistry using DAB, you require a secondary antibody that is conjugated to horseradish peroxidase (HRP). The HRP catalyzes the conversion of the DAB substrate into a colored product. So, if you do not quench the endogenous peroxidase activity within your cells, then you run the risk of non-specific staining (i.e., a false positive). I suggest incubating your cells in 0.1% hydrogen peroxide (in 1x PBS) for 10 minutes after you have washed your fixative off of your cells. Alternatively, you could run an additional control in your experiment whereby you do not undergo a hydrogen peroxide step to determine the effects on your staining. Good luck!
Depends on the type of cells you are staining. endogenous peroxidase is expressed in myeloid cells predominately. If you are using a different type of cells it may not be necessary and hydrogen peroxide can be damaging to your cells.
Erica gave you a very good "general purpose" answer, fitting both for IHC on tissues and on cells. For tissues - peroxidase blocking is essential, because in every tissue there are cells containing peroxidases (if the tissue is vascularised). For cells - it depends what type of cells you are working on - as you may not need a peroxidase blocking step if your cells have only negligible amount of endogenous peroxidases. Lisa is right that hydrogen peroxide may be damaging to your cells. That is true for all tissues and cells - but there is a way around it. There is information in the literature that there are antigens affected by hydrogen peroxide step (e.g. lymphocyte markers CD4 & CD8) to the point that staining is very weak. In such cases the peroxidase blocking step needs to be performed later in the procedure, just before the reagent conjugated to exogenous peroxidase (HRP). Obviously, in such situation you couldn't use primary antibody-HRP conjugated because the antigen would be damaged, but if you were using unconjugated primary antibody and biotinylated seconday antibody followed by avidin (or streptavidin)-HRP, then your blocking should be just before the last step (avidin-HRP). This way peroxide would not damage the antigen in your cells/tissue and primary and secondary antibody binding. For most of the antigens the damage to the antigen is negligible and the immunostaining is good enough for assessment, but for very labile antigens moving the peroxidase blocking step to just before reagent bound to HRP is essential.
You haven’t specified what cells you use or what antigen you want to disclose - but for the sake of your project you could do 3 parallel experiments: 1). IHC without peroxidase block, 2). block after cells permeabilisation, 3). Block just before reagent conjugated to HRP. Good luck with your work.
thank you very much Lisa and Maria for your answer and well explanations , I am working on primary chicken kidney and MSB1 cells ( Lymphocytes infected with MDV)
In our institution, we use inespecific peroxidase blocking systematically. In avidin-biotin-peroxidase-DAB IHC, the results for micrography are better.
thanks Jose Silva so you advice me to use the peroxidase blocking systematically with any type of cells or tissues
Hi Maria,
Thanks for your very detailed explanations. However, I cannot figure out that you said "but for very labile antigens moving the peroxidase blocking step to just before reagent bound to HRP is essential."
Does it mean that peroxidase blocking for the very labile antigens is needed but should be pay more attention (e.g. H2O2 concentration and treating time) just before the addition of HRP?
Thanks and regards,
David
Dear David,
Sorry, I had tried to be clear, it seems that I didn't manage :) What I meant was that for primary antibodies to certain antigens (as e.g. those quoted above) it is advisable to perform the endogenous peroxidase blocking step AFTER incubation with primary antibody and preferably even after secondary antibody (biotinylated), just before Streptavidin-HRP. This is to ensure that there is no damage by hydrogen peroxide to the tissue containing an easily damaged antigen. The blocking needs to be only before the step containing HRP tag, which is, after reaction with a chromogen, an indicator of the presence of antigen. I hope that I have unscrambled my previous answer. Good luck with your experiments.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA