In the past, I have used freshly-made 4% paraformaldehyde for transcardial perfusion of mice/rats and subsequent post-fixation of whole brains. However, for practical reasons I am interested in replacing freshly-made PFA with a premade formalin solution. From what I understand, 10% formalin and 4% PFA are equivalent, with the exception that premade formalin typically has methanol added as a stabilizing agent. Anecdotally, most researchers I know use fresh PFA, and in researching this issue, it seems that the reason is to avoid methanol. However, my IHC protocol calls for H2O2 in methanol to quench endogenous peroxidase activity. So, I'm having trouble reconciling these. Can anyone provide info as to why formalin with methanol is undesirable for tissue fixation, and what are the real consequences on using it for later IHC? Thank you, in advance!
To provide some relevant details, my interest is in whole rat brain, to be cryopreserved and thin sectioned for single and double immunohistochemistry (DAB and/or fluorescence) for NMDA receptors, immediate early gene activation, scaffolding proteins, etc. on free-floating sections.
In Routine histology we use 4-8% Formaldehyd phosphate-buffered and made from 40% formaldehyde stocksolution with small amount of Methanol.
We do regularly IHC for a big amount of antibodies on tissue fixed between few hours and few days. So in my opinion NBF is highly suitable for fixation and following IHC.
I would hope, that someone really can give first-hand infomation about negative influence of the small part of methanol on a list of antigens. For me it seems like an urban legend, that goes back to the times, when fixation at all was seen as "no-go" for IHC.
So the point is still: Trial and error. If it works, then it is ok.
Dear Katherine,
It's not clear to me whether you already have a protocol that works for the antibodies you mentioned, or you are still in the preparation phase, setting up the experiment based on your SOP.
Whether methanol will interfere with your staining or not mostly depends on the fragility of the antigen, and your antibody, (polyclonal vs monoclonal, sensitivity of the exact epitope to methanol, etc).
It would be also interesting to know when you do the H2O2 blocking: if it takes place after the primary antibody binding, then it won't disturb the antigen recognition, while in an earlier step (e.g. during fixation) it might.
So, bottom line: if you already use a protocol that works with H2O2 in methanol before the primary antibody, then go ahead and simply change PFA to formalin. If you haven't done these experiments, then it will come down to testing the two fixation methods. What I can tell you with much certainty is that the H2O2 blocking before DAB staining doesn't have to happen in methanol, you can also use PBS, we do too. This way if methanol does turn out to be an issue for some of your antigen/antibody pairs, you can always change it to PBS, while stickig to 4% PFA.
In Routine histology we use 4-8% Formaldehyd phosphate-buffered and made from 40% formaldehyde stocksolution with small amount of Methanol.
We do regularly IHC for a big amount of antibodies on tissue fixed between few hours and few days. So in my opinion NBF is highly suitable for fixation and following IHC.
I would hope, that someone really can give first-hand infomation about negative influence of the small part of methanol on a list of antigens. For me it seems like an urban legend, that goes back to the times, when fixation at all was seen as "no-go" for IHC.
So the point is still: Trial and error. If it works, then it is ok.
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