I have been successfully labeling thyroxine in the thyroid follicles of whole larval fish using a primary antibody specific for T4 (1:500) and AlexaFluor 488 as a secondary antibody (1:150). I have done this successfully several times in several groups of fish. However, when I returned to this method to process additional samples (~1 year later) I found that the labeling appeared incomplete and blotchy. As it appears as though rings are being formed around each follicle with greatly diminished staining inside the structure. Given that the methods have not changed and the age and size of the fish is also the same, I suspect that one of the reagents that I am using has expired or is no longer working.
Example images may be found below.
Any insight into what may be happening would be extremely helpful.
It must have something to do with your antigen/tissue; expired antibodies would give you less signal but they won't change their target antigen.
Did you perform the staining on sections that were stored for one year? If yes: How did you fix the sections and how were they stored? If the proteins are not properly fixed and/or the storage conditions were too warm, the antigens might have diffused.
Hi Vivica,
Thank you for your answer. I also considered this possibility, but this has also been happening with "fresh" samples as well (less than a few months old). We fix each larvae by incubating it in 4% Paraformaldehyde overnight at 4C. The next day we dehydrate the samples using gradually increasing concentrations of MeOH and storing at -20C in 100% MeOH.
Is there a reason why you store it in methanol and don't complete dry the sections followed by rehydration when incubated? As far as I know methanol is not only unhealthy but also hygroscopic. Maybe it takes up water over time which ruins your section?
The paraformaldehyd might also be washed out very slowly.
Hello Leah,
I am also trying to do this staining procedure on larval fish (fixation in PFA O/N at 4°, conservation in MetOH) but for the moment I failed obtaining good results. Did you do this staining on all the body of your larvae? or did you do it on larval sections ? what are the standard length of your larvae ?
Thank you very much for your answer.
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