Hi there-- I would stick with the 3% BSA in PBS (I have the most success with this)... make sure it is freshly made-- anything growing in there will have drastic effects on your staining. Your concentration of triton is mild (which is good!) for cell culture. Personally, I usually use .1% saponin for cell culture permeabilization. I also use a stock of the permeabilization solution for all washes and even perform the perm and blocking step at the same time for 1 hr (just add your blocking agent, BSA or serum, to the permeabilization stock at the right concentration). This will save you a step in the procedure. For my stainings, I continue to use the mixed perm/blocking solution for primary and secondary antibody incubation. Wash with perm solution and do a final wash with PBS before mounting. I also typically use a 1:500 dilution for the secondary antibodies (though yours does seem to work at 1:1000). You will want to avoid freeze/thaw cycles with your antibody, so I recommended you aliquot (~10uL) into PCR tubes or other tubes so you can access small amounts without thawing your whole stock at once. If you want to speed up your procedure, 1hr primary antibody incubation at room temperature should be sufficient. I hope this helps! Good luck =)

Hi your consecutive images look like autofluorescence or background noise. I would try this recipe for permeabilization and blocking. 30 ml PBS, 60ul Tween, 1500ul Serum, 60ul Triton. We do this in one step for 45 minutes. Doesn't seem like your permeabilization is quite long enough and you may need the addition of Tween.

Usually the combination of the 2 works well for us. Goodluck.

Hi Bertha Mak

There maybe several things going on here.

1. Usually to visualize intra-nuclear proteins (not bound to nuclear membrane), I recommend acetone-methanol (1:1) fixation which precipitates all the proteins thereby increasing epitope availability

2. Mixing sera for blocking is a bit tricky. What species is the secondary antibody raised in?

3. As suggested earlier, repeated thawing freezing cycles is a bi no-no, but I doubt that was the issue here.

4. Do you also run secondary only controls to check for cross-reactivity?

Best

Aparajita

Aparajita Lahree Hello

1. I have another experiment before which I need to stain the same nuclear protein here but it is another antibody (not the same one with this post). I used 4% PFA in PBS for 10 mins (same as this one) and it worked well (the protein is really "nuclear"). So I was thinking like maybe the same fixation works with this Ab as well as they both target the same protein. I have never consider another fixation method before but maybe I should try it.

I am going to do co-localization analysis here and is it an issue if I change the fixation method to methanl-acetone fixation? I searched briefly and I learnt that methanol-acetone fix the cell by denaturation and will it disrupt the protein binding here as well (i.e is methanol-acetone fixation a good choice for localisation studies, calculation of pearson's correlation etc)

2. Donkey. I tried anti-donkey serum in another trial but it looks the same.

3. good to hear that. If it is the problem of the primary Ab guess I don't have any solution here apart from buying another one.

4. Yes. I tried to stain the samples with 1:1000 anti-rabbit antibody. Very little signal observed but I think it is acceptable and won't interfere with the primary staining.

Updates: I changed the secondary antibody to another wavelength (ie also anti-rabbit but in red). It does seems to be better so I am guessing maybe it is the problem of the secondary antibody. Although it looks better but a lot of times I started to get images which shows the protein is in cytoplasm while very little is in nucleus. It was a bit terrifying as this is a well-established nuclear protein (?). I am glad that it looks a bit better tho, still on the way.

thanks for all of your help :)