We are trying double IHC staining with 2 mouse IgG1 monoclonal primaries. In both cases the chromogens (Fast Red & Vector Blue) bind to AP and the secondaries in both reactions are AP-labelled proprietary polymers. We are getting cross-reaction, likely due to 'hangover' AP-labelled polymer.
Any ideas how to quench this before the second reaction?
Do you perform a HIER step in between the protocols? the high temperature should denature the enzyme.
Thanks for your reply. No the HIER step is before the 2 sequential reactions. In between there are just washing steps and no further retrieval.
Chris, you could try with two different labels, means HRP for the first antibody (preverably diaminobenzidine), then AP secondly.
Thanks to both. We will try HRP then AP, the difficulty has been that the best chromogen combination was with two AP-linked chromogens (we are trying to deconvolute colours without the advantage of spectral separation). MOM kit? I will investigate.
If you can find one, you can use either a tight binding or irreversible inhibitor of AP. There is one inhibitor I found in the literature. It is 2-( phenylsulfonyl) oxidase for tyrosine phosphatases. You can try this technique if you absolutely have to use two AP groups. But I would go with the HRP and AP., myself per Torsten's idea.
Sorry, the spell check changed my answer. It is oxirane and not oxidase.
IHC method to look at two antigens has its own technical short commings. like dye precipitate of one enz interferes with the efficiency of other, overlapping precipitations, signal seperation requiring sophesticated imaging tools, reproducibility etc.
IF with two different abs is your best option.
Good (usable) reagents = good data
Depending on which advice listed above you choose to follow - here is some additional information:
You can achieve a very good colour separation using AP and HRP and choosing substrates (chromogens) with contrasting colours. You didn't specify which colour combination worked best with your colour separation using your particular software, but you can find the same colours searching available substrates from leading manufacturers.
The best red chromogen for AP I have ever used was New Fuchsin, but next in line is blue chromogen. You can complement those with a wide range of chromogens for HRP (e.g. see ImPact Peroxidase substrates from Vector Labs). Or you can use blue chromogen for AP and NovaRED from Vector: DAB, AEC,VIP,SG). You can modify DAB colour with nickel or cobalt salts and achieve much darker brown-black permanent staining. There is a fair amount of chomogens on the market from almost any company catering for IHC, but you meed to be careful and check that your final step (dehydrating and mounting) is not diminishing the staining (happens if the chromogen is soluble in organic solvents) and not all chromogen kits using the same substrate produce the same 'colour fastness' - in such instance you need to use aqueous mountant e.g. Dako Ultramount, which has an additional advantage of solidifying).
One of the oldest methods to block alkaline phosphatase for IHC is using Levamisole.
I agree with Suresh that the safest and the least time consuming method would be to use antibodies with two immunofluorescent tags.
Sorry, you did include your preferred colour range - my mistake. Good lick with your experiments.
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