Hi Edna,

one possibility why you have problems with the detection from the directly conjugated antibody (mouse) is the treatment with HCl for the BrDU-staining. The problem here is not that you are using a directly conjucated antibody but that HCl might destroy the antigen epitope that is recognized by this antibody.

However, HCl will not destroy all epitopes recognized by antibodies so it can very well be that one of your antibodies is still working fine (as it seem the case in your experiment) and the other will not work properly. I am not sure about you protocol but do you use a borate buffer after treatment with HCl to neutralize remaining acid?

I also found a paper with a different (a little bit exotic) method for BrDU staining followed by IHC with additional antibodies. It seems to be some kind of heat treatment (I also used heat treament with some antibodies - sometimes this works but not all the time). This is the titel of the paper:

"Antigen-Retrieval Procedure for Bromodeoxyuridine Immunolabeling with Concurrent Labeling of Nuclear DNA and Antigens Damaged by HCl Pretreatment - PMC"

Maybe this helps - good luck

Hi Dörthe,

Thank you for your suggestions!

It's true that I am not using any neutralization buffer for HCL, since it has always been working just like it is before.

I also thought about the HCL being the problem. In the papers that I found using a non-conjugated Prox1 antibody together with BrdU, there never seemed to be a problem in staining with HCL. Some of them neutralized with Borat buffer and others did not.

I am also going to have a look at the paper you sent!

Do you think I could try a background quencher like Trueblack together with longer blocking? Or should I try a shorter HCL incubation?

Thank you!

Hi Edna,

Regarding your question - think it is worth to give both a try - maybe this is already helping.

And try the neutralization with borate buffer - even if the Prox-1 antibody works good with BrDU and the HCl treatment, this must not be the case with other antibodies, as this depends strongy on the epitope recognized by the antibody. Maybe with your antibody borate buffer treatment can improve results.

I know it can sometimes be difficult to do a double or triple staining and sometimes it is little bit of "try and error" to figure out the conditions to get good results with all antibodies together.

Good luck!

Edna,

Some years back, I developed an immunostaining protocol that allowed staining for BrdU and other things. The HCl step was after staining for the other protein, which normally would knock off the antibodies. But, I used the covalent crosslinker DTSSP to link the first primary and secondary antibodies in place before HCl treatment. Then came the HCl treatment and immunostaining for BrdU. It worked well. I also did it this way because 2 N HCl certainly might denature some epitopes on proteins and make staining for them weaker or not possible. Here is the reference:

Spatiotemporal gradient of oligodendrocyte differentiation in chick optic tectum requires brain integrity and cell-cell interactions. Glia. 2003 Jan;41(1):25-37. doi: 10.1002/glia.10163. PMID: 12465043.

Best,

Deni