Both rabbit (anti-Sox2) and mouse (anti-PCNA) primary antibodies work in single IHC but not in a double IHC. This is true for a simple mix of both antibodies as well as a sequential IHC. This also occurs with different combinations.
Has anyone encountered this or has a solution?
Details:
- 4% PFA/sucrose adult mouse brain tissue (16 um)
- citric acid + microwave treatment for antigen retrieval
- 5% NDS + 0.2% Triton-X in PBS / 4% FCS in THZT
- rabbit anti-Sox2 (1:500), mouse anti-PCNA (1:500)
- donkey anti-rabbit Alexa Fluor-488, goat anti-mouse Alexa Fluor-594
Hej, could be some unknown binding/interference of the two primary antibodies. It is pretty much a black box so who knows. I have experienced such things very rarely as well. You could use Tyramide amplification and brake it down into two steps with stripping the first antibody in between.
Dear Swip Draijer ,
1-Please reduce the concentration of your PCNA Ab to 1/1000, it has a very high intensity signal which has overlap with another antibody
2- Use the matched serum with your secondary antibody
3- brain tissue has a very high auto-florescence before doing any step leave your slides behind the window to get some sun beam it will reduce background staining.
4- overnight incubation with primary Ab should be in cold room and with secondary should be in RT for one hour.
hope this helps,
Good Luck,
Masoumeh
Dear Masoumeh,
Thank you for your answer, I will take your advice into account. I do use overnight incubation at 4 degrees with primary Ab and 1 hour incubation at RT for secondary Ab.
Whereas these steps would reduce background, I do not think this is causing the failed double immunohistochemical stains since the single IHC work fine without much background.
Thanks,
Swip
Hej, could be some unknown binding/interference of the two primary antibodies. It is pretty much a black box so who knows. I have experienced such things very rarely as well. You could use Tyramide amplification and brake it down into two steps with stripping the first antibody in between.
How do you know that the staining didn't work? Was there absolutely no positive staining, just less signal, or higher background?
Do you keep the NDS and FCS throughout the primary incubation? If it were me I would test different blocking material, such as 1-2% BSA to see if it is something in the NDS. Is there a reason why you are using both NDS and FCS? When you do sequential, have you checked to make sure that the first antibody set worked as expected?
HI, all.
When you do IHC or IF in mouse samples with a primary antibody raised in mouse and It is a complete IgG, for instance, It may be arrested in tissue by its Fc and so, It will not be detected, at least partially, by the secondary because it recognizes the Fc portion (If so).
So, I guess your problem will need some FCR blocking in the first steps. You can use anti-FcRII and III ( anti CD16/32) or double NDS and NMS (Normal Mice serum) to block it.
Hope it helps.
I don't know exactely where the antigens are localized but maybe you witness sterical problems when combining the antibodies? This might explain why it even does not work when incubating sequentially. When incubating sequentially: Do you incubate the two primary antibodies before the secondaries? Especially after binding of the secondary antibody there might be sterical hindrance.
Hi, I got the same issue recently. The 2 antibodies are perfect in the IHC, however, when I apply the dual stain did not work. I tried different concentrations and overnight. Also, I have not combined them. I am trying to optimise them separately.Any suggestions.
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