I used an Alexa 488 anti-rabbit secondary antibody for colon tissue samples (that had gone through FFPE and rehydrated, followed by antigen retrieval) but the stain was really weak and the signal was practically undetectable. Stains for Beta-catenin and ALDH1A1 worked very well. One of my coworkers suggested next time using an anti-rabbit HRP conjugated antibody and then performing tyramide amplification to improve the signal. Since I'm trying to conserve time and tissue, I was wondering if it's possible to re-incubate my slides in fresh secondary and then re-mount them. Thank you!
Hi Jess,
You can definitely try re-staining the tissue, however, make sure (check the data-sheet) you are within the recommended dilution range. Perhaps add a more concentrated amount for a stronger signal? I would recommend only re-staining the tissue once or twice. If you have spare tissue, compare both methods (high concentration OR HRP) to compare results. Hope this helps!
I actually just washed the slides ~4X in PBST, then re-incubated in the HRP-conjugated secondary and performed the downstream tyramide amplification and the stain worked beautifully with nothing showing up in my isotype or no primary controls. Thank you so much for your help!
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