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fluorescent immunohistochemistry problems - (Sep/26/2013 )

Hi everybody!


This is my first post on this website. I hope you can help my with a problem.


We was established the optimal conditions for the immunohistochemistry using fluorescence against a transcription factor in paraffin embedded tissue. Suddenly, without any changes in the protocol, the staining didn't works properly, we can't detect any staining often and we we have staining, it is dirty and weak. We are using microwave-mediated antigen retrieval in citrate buffer and HRP-conjugated secondary antibody with amplification using TSA system. Tissue is paraformaldehyde fixed and paraffin embedded. We have already change all the buffers, the primary and secondary antibodies, the TSA system, we check different tissues positives for our protein and we are success randomly. Some months ago the staining worked in the 99% of the cases. Maybe some of you have had a similar problem and could help me? I ran out without ideas that what could be happening!! unsure.png


Thanks in advance!


It could be quite a number of things, could you provide a working and non-working picture?


The most likely scenario is that one of the reagents has gone off - this is likely to be either the primary or secondary antibody.

Occasionally fixatives degrade over time, and this will result in your IHC not working well with increased background.


Another scenario is that one of the buffers you are using has been prepared incorrectly, so it may have the wrong pH or too much/little salt.


although not as likely, your retrieval may not be working as well as in the past. it's possible that your microwave oven may be failing or you're putting your sample in a different place in the oven.


have you tried a traditional method for antigen retrieval?



Thank you for your answers!

Bob1, thank you for the advices. We have already changed the antibodies and we prepare fresh paraformaldehyde each time, cos we use comercial fixative.

About the buffers, we have changed all several times but maybe there is one detail that we didn't realize yet that is wrong. I will provide a picture soon! Thank you very much


mdfenko, It's interesting the fact you propose. The microwave is the same that one year ago, is it posible that the power of the mcirowave becomes lower with the time? This antigen takes more time that others to be retrieved, and it's true that the "normal" antiges sometimes now works worse than in the past... Do you think that the microwave power it such a critical fact in the protocol?

what do you mean with "traditional method"? Enzimatic antigen retrieval or pressure cooker? Thank you very much for your help!


microwave power can, indeed, make a difference in retrieval. it will influence necessary time of exposure to microwaves.


traditional methods would be what was done before anyone tried using a microwave oven to speed up the procedure.


we used xylenes to remove paraffin then decreasing concentrations of ethanol to rehydrate the tissue.