does anybody have experience on immunofluorescent staining of FFPE tissues for LMD? Need to perform a triple immunostaining of breast tumor tissue for subsequent isolation of tumor-infiltrating lymphocytes and DNA analysis. The staining protocol works perfectly on the glass slides, but antigen retrieval step seems to be very critical in the case of membrane slides. I have to perform HIER with Tris-EDTA at 95°C for 20min for CD4 and Foxp3 stainings. My LMD slides are PPS metal framed, the membrane withstands the heat, but the tissue doesn't stay on the membrane. Do you know some tricks how to increase the adhesion of the tissue to the membrane (already tried UV irradiation)? Or is there some good alternative AR protocol for these particular antigens? Already tried prolonged incubation with retrieval solution (16h, 24h of 48h at 60°C; 40min at 80°C), proteinase K and trypsin - none of those work.
Another problem that I encountered is a very strong background of my stainings and poor morphology (both for immunofluorescence and DAB). Are there some critical steps in the protocol that I should consider (for example, concentration of antibodies or incubation times should be dramatically different from stainings on the glass slides).
Is there any way to improve the morphology of the slides?
I would very much appreciate any advice.