Two things you might try (if you haven't tried them already):

1) Have you tried putting orthovanadate in the fixative to preserve the phosphorylation of the S6 protein? If the endogenous phosphatases in the tissue aren't sufficiently inhibited, they could be removing the phosphates from the protein resulting in a loss of signal.

2) You might try antigen retrieval on some of the 24 hr fixed tissue to see if it restores your PS-6 labeling. We find that using a hot citrate retrieval step is helpful for many antigens.

Hi David,

I have tried the antigen retrieval, I boiled the tissue for 5 minutes in citric acid before staining. Unfortunately this did not elicit any PS6 staining. Interestingly enough, when I tried this on the vasotocin stain for consistency it also seemed to have somewhat of a negative impact on the staining.

I will look into the orthovanadate though, I think this may be a step worth considering! Thank you!

In case its of any use to you, I've attached the protocols we use for immunolabeling frozen and paraffin sections (we work mostly in brain and retina), which include what we do for antigen retrieval (including the citrate buffer formulation we use). A big difference between what we do and what you tried for antigen retrieval is the temperature and time of the citrate retrieval step. I don't know if this would make a difference.

We never expose the sections to boiling citrate buffer. We heat the buffer up to 90-95 degrees C and then immerse them in the citrate. We then let them stand at room temp on the countertop for 30 to 60 min. I've never tried a real short incubation in the citrate buffer. 5 min may not be enough time to get the full benefit.

Dave Sherry

I will definitely give these a read, thank you! I have also recently seen an antigen retrieval protocol where hot citrate was pipetted onto slides for 5 minute incubations 3 times, I may try a few different variations of this including the method that you use. If there's a method that can bring back expression of the PS6 protein while not negatively impacting the vasotocin expression then that would definitely make a lot of things possible!

Thank you!

Hi David, quick follow-up; I think that this orthovanadate step will be a very good one to test in order to see if it's the paraformaldehyde or the phosphorases that are inhibiting the signal. Do you have a personally recommended concentration for using it at?

Hi Jeffrey,

would it be possible for you to use a different fixation? If one of the epitopes is so sensitive maybe an alternative fixation with eg. acetone might be an alternative which could work for both. We had to switch to acetone for some antibodies in cell culture experiments due to epitope masking by PFA. In tissue we recently successfully used antigen retrieval with 110°C for 10 minutes. But in my opinion boiling tissue should be the last possibility since the tissue quality is suffering.

Good luck.

Best, Claus

Hi Claus,

It might be worth looking into alternative forms of fixation. I'll have to do some reading about how different fixation techniques may impact antigens in zebrafish tissue as they do seem to be a bit more fragile than the rat tissue I have previously worked with. I will see if others are using similar fixative techniques, because as you said, it is entirely possible that the PFA is just not going to react well with the target proteins.

As far as the previous suggestion with using orthovanadate in the fixative, I do seem to be having some difficulties finding studies where any groups have actually used it in the fixative for immunohistochemistry, rather using it as a phosphotase inhibitor in homogenization solutions for immunoblotting. If it is something that would work in PFA that would also definitely be something I would try.

Use of microwave can also help with antigen retrieval in paraffin so maybe microwave treatment of slides.

Hi Leonie, yes this is something we have tried. Today I am going to try implementing David's suggestion of heating the citrate buffer and incubating the slides in the heated buffer as it cools for ~30-60 mins to see if this works, as our initial attempt of boiling the slides in citric acid in the microwave did not elicit any change in antigen expression. We have also added orthovanadate to the fixative both in post-fixed whole brains and brains sectioned onto slides in order to try to quench endogenous phosphotases that may be breaking down the target protein.

Just to add an update for future reference if anyone else has a similar question;

We were unable to achieve staining using the PS6 antibody in overnight fixed tissue. We added 10mM sodium orthovanadate solution to 8% paraformaldehyde to make a 4% solution including 10mM orthovanadate. We post-fixed both whole brains and slide-mounted brains in this fixative overnight in order to determine if the type of fixation would make any difference as well. Additionally, we performed antigen retrieval on half of the slides using David's protocol (heating the citrate buffer to ~95C and then incubating the brains in this warmed buffer for 60 minutes).

Unfortunately none of these methods were sufficient to obtain PS6 staining using fixed tissue. Interestingly enough, this method of antigen retrieval also quenched vasotocin and isotocin expression, which we now find to only work in fixed tissue that has not undergone antigen retrieval.

Thank you all for your help, but as of now it would appear that the double-label staining is something I will have to set to the side for now.