I’m trying double-staining for co-localization of cFos (alexa 488) and TRPV1 (alexa 594), the main problem is that my fluorescence is too weak and I have these antibody conglomerates. As it can be observed, they’re worst for alexa594, also, they were unusual before I introduced an antigen retrieval step. I also worry because these conglomerates are not just on the tissue but also around them.
Fixation PFA 4%
Antigen retrieval: citrate buffer 70ºC 1h
Blocking: 20 min Glycine + 1h BSA
Primary antibody 1:100 72h incubation in BSA+triton
Secondary antibody 1:500 2h
Mounting medium: Fluoromount G
I tried to decrease antibody concentration, incubation time and I also tried without antigen retrieval, but the consequence is always less and less fluorescence without avoiding the background and noise.
You could try to briefly spin the antibody vials before pipetting and then take liquid from the top.
*Edit: Since the background fluorescence seems to be worse in the green channel, you could try to switch this secondary antibody more to the far-red (e.g. Alexa 649), if your microscope has suitable filters.
Hallo Lia, I agree with Theresas recommandation. These clumps, you are showing, can also come from the BSA. Filter BSA before using it, especially when you prepare it freshly from powder.
Instead of BSA you can try TBS or PBS with 1% normal sera and 0,1%Troton-X.
For the Blocking step you can try 10% n-Serum in 0,2% TX-PBS or TBS. You do not need glycine for c-fos, for the other ab I don't know. but if it is not nacessary, scip it. Bevor you start with your double labeling, try to find out in a single way what kind of tratment each antibodies need and then put them together.. Good luck!
The above suggestions from Theresa and Neubacher are good. I'd agree with switching to normal serum for your blocking/antibody buffer (of the same species of your secondary antibody) if the BSA is causing problems. I've used 10% serum in-place of 1/60+BSA plenty of times.
I think you may benefit from playing around with the antigen retrieval if you're not seeing much signal. In our lab we typically use 10 minutes at 99'C in citrate buffer, heated in a microwave (or waterbath, but you'll need longer to bring it to temperature). But you have to be careful with brain sections when using a microwave, I've had bubbles forming at the ventricle that can lift the hippocampus off the glass.
Keeping your primary antibody at 4'C instead of room temperature can also help with specificity, especially if you're doing incubations in excess of an hour or two.
do a control with 2ndary antibody only (skip primary). you will likely see the same clumps. Check how old your primary and secondary antibodies are, and what are their storage conditions. The antibodies may be old. The 2ndary antibodies could be diluted too quickly (clumps are present). Use your 2ndary antibodies on sections, which never had such problems in your/other people hands. These controls will help you narrow down the problem (most likely problem: 2ndary antibodies are old or insufficiently diluted, with clumps present)
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