Dear Aishah,

please follow this,

How to block endogenous peroxidase activity on frozen sections

Immerse slides in fresh made 0.3% hydrogen peroxide in 0.1% sodium azide for 10-15 minutes (to make the blocking solution, add 5ml of 3% hydrogen peroxide to 45 ml of 0.1% sodium azide and mix well). An alternative is to use 0.3% hydrogen peroxide in methanol for 20-30 minutes since methanol accelerates the destruction of the heme groups so a lower concentration of hydrogen peroxide can be used for longer incubation time.

you could find best tips in this website:

http://www.ihcworld.com/_technical_tips/peroxidase_tips.htm

Hello,

when performing immunofluorescence you never have to block endogenous peroxidases!

However, if you want to finally visualize your immunostainings with an ABC-complex and diaminobenzidine you allways have to block peroxidases (on paraffin or frozen sections).

For example you incubate your slices in 3% H2O2 solved in PBS for About 15 min at room temperature.

Kind regards

Stefan

It would depend on your chosen method, any methods that rely on Horseradish Peroxidase (HRP) mediated reactions could be affected by endogenous peroxidase.

So if you are using a directly conjugated fluorescent antibody or alkaline phosphatase mediated reactions, it is not necessary.

If you are using HRP then you may be able to leave it out and still get good results, it is dependent on the tissues being used.

It is probably best to do a run to compare with and without, if you have the resources.

Absolutely no needs this step.

Hi ! Just adding here from my own recent experience in IF on paraffin sections - There were observed differences in staining when not blocking peroxidase in paraffin sections for IF versus when done. Shortly , I would recommend to block it anyways just to get lesser background and better staining .