Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Inhibiting endogenous peroxidase
1. What is the best way to inhibit endogenous peroxidase activity before doing an immunohistochemical
2. How long can methanol/H2O2 mixture (for quenching endogenous peroxidases during IHC) be kept? or
should it be freshly made each time before use?
Different people favour different methods! Here are five suggestions. All are claimed to work well, so probably you should start with whatever you think is the easiest and cheapest.
We use a homemade version: PBS with 0.03% hydrogen peroxide, and 0.1% sodium azide. Very gentle; doesn't knock sections off slides (frozens); can make up a one-week supply. Use it once, then discard (we use dropper bottles). Our PBS is at pH 7.4. We collect the leftover for chemical disposal of sodium azide.
OR you can purchase DAKO peroxidase blocker with 0.03% H2O2. This block works best with our mouse antibodies as it does not interfere with some of the IHC staining/per recommendation of PharminGen. They use DAKO also, and if there are capillary gaps involved, this does not produce the crummy bubbles that drive one crazy.
We prepare 600ml vats of methanol/H2O2 for use on a DRS601 and replace these weekly. It's left on the machine for 5 working days then dumped. We're handling about 150 ICC slides/day.
Just a personal note on the use of methanol in blocking solutions; I have also found that methonal can be harmful to some antigens, both hemopoetic and some infectious disease antigens. We have found that performing our endogenous peroxidase inactivation prior to any antigen retreival step (either enzyme digestion or heat induced) works best. For antigens sensitive to methanol and frozen sections we use PBS containing 0.1% Na azide and 0.5% H2O2 with excellent results. Just be sure to wash the slides well after this step because the Na azide is a potent peroxidase inhibitor which will eliminate any specific staining quite well. Using poly lysine coated slides will generally keep frozen sections from lifting off.
Brian J. Chelack
Quenching with the glucose oxidase method works very well, and is very gentle on sections, particularly frozen sections. The only drawback is a bit more preparation of solutions, but in the long run is a very COMPLETE quenching, better than hydrogen peroxide, according the original publication and method. I highly recommend it.
Complete inhibition of endogenous peroxidase (including activity in leukocytes and erythrocytes) can be achieved
by treating formaldehyde- or acetone- fixed smears or sections with 0.024 M hydrochloric acid in ethanol for 10 minutes. To make this, add 0.02 ml of concentrated (12 M) hydrochloric acid to 100 ml of ethyl alcohol.
Weir EE + 4 others (1974) Destruction of endogenous peroxidase activity in order to locate antigens by peroxidase-labeled antibodies. J Histochem Cytochem 22:51-54.
This simple method doesn't seem to be much used. I have tried it, and Yes, it did work.