I'm performing immunofluorescence histochemistry on tissue sections and targeting two antigens that require different antigen retrieval methods—one requires enzymatic digestion (proteinase), while the other needs heat-induced epitope retrieval (HIER) using Tris-EDTA buffer at pH 9.0 (125°C for 15 minutes). How can I optimize my protocol to preserve both antigens effectively? I'm using the Olympus FV3000 confocal microscope for imaging.
Many Abs only give signal after a certain antigen retrieval (AR) step on FFPE material. Various strategies can be used to perform that retrieval step. Every lab has its own ways to come to an optimal result. When such an optimization is performed one will find that method 1 gives poor results, a 2nd method gives a 2+ staining while the 3th method gives a 3+ staining.
It may be that your Ab can be used after 1 of the two AR-steps you describe, so you have to try that.
Otherwise you may want to combine the methods. The you have to try HIER first and perform the protease step next, followed by the Ab-incubations. If that doesn’t work try the other way around.
If that doesn’t work you will have to re-evaluate your sample procedure: do you really need fixation at all? Can u use frozen tissue, etc etc.
Good luck!
DK
Duco Kramer Thank you so much for your recommendation. I will try each one first, starting with HIER, before combining both methods.
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