We have mouse paws that were intended for other uses and stored in 70 or 100% ethanol for a year. What are my chances of being able to post-fix in formalin, then paraffin embed/section, to perform IHC (likely more problematic) or more conventional staining to examine cartilage and bone? Will antigens be retrievable? Will cellular morphology be intact? Also, for an EDTA decalcification of such small samples, do we need to remove soft tissue first?
As with everything in IHC, you will have to try and see. Some IHC antigens and protocols are more forgiving than others. I would suggest processing a couple of samples and comparing the IHC results for your markers of interest with results from standard processing, only this will tell you if you have changes in antigen detection. In general 70% ethanol is fairly forgiving, but if your tissues went straight to ethanol (without a prior formalin fixation), they might now be quite dry and brittle, and histology might not be great (very eosinophilic). I'm not sure what 100% ethanol will have done for a year, nothing good I would expect.
As with everything in IHC, you will have to try and see. Some IHC antigens and protocols are more forgiving than others. I would suggest processing a couple of samples and comparing the IHC results for your markers of interest with results from standard processing, only this will tell you if you have changes in antigen detection. In general 70% ethanol is fairly forgiving, but if your tissues went straight to ethanol (without a prior formalin fixation), they might now be quite dry and brittle, and histology might not be great (very eosinophilic). I'm not sure what 100% ethanol will have done for a year, nothing good I would expect.
Seconding Jean-Martin's opinion, adding:
As long as one does not know anything about the REAL processing PRIOR to the described storage of the tissue 'in 70% or 100% ethanol [the latter equal to a "rude dehydrating and precipitating" alcoholic fixation, if primary fixation wasn't done with a more sophisticated alcoholic fixative like Carnoy's or Methacarn, other alcoholic fixatives with commercial (trade mark, trivial) names etc.....] there will be no (theoretical) help even if it can be only an extempore technical consideration.
Perhaps (only one example out of many) you may read:
HOWAT & WILSON: "Tissue fixation and the effect of molecular fixatives on downstream staining procedures" in Methods. 2014 Nov; 70(1): 12–19. @ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240801/ , e-pub (beta): @ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240801/epub/
Moreover, if you think about a 're-hydration' followed by "classical fixation" of the stored tissue(s), this will not help you with an improved or, ultimately, an 'intact' morphological appearance or even an improved reactivity (with regard to IHC....) and it is to expect that there is no difference using EDTA or nitric acid for decalcifying your tissue (dividing your sample of mouse paws - if they are calcified considerably at least once for exposing "bone proper" to the decalcifying agent), to assess the calcification level see also: https://www.researchgate.net/figure/51712640_fig1_Radiographs-of-interphalangeal-joints-from-paws-of-wild-type-versus-ankank-mice-We-show (radiographs for the wild type aged - 8 - 12 - 18 weeks). Best wishes and good luck....!
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