Hello everyone,
We are struggling with fluorescence immunostaining of paraffin-embedded sections of TA muscle. We're staining with laminin, pax7 and eMHC.
When we first stained I used the pressure cooker antigen retrieval method (Citrate buffer), in this method PAX 7 and eMHC worked well but we had no signal from the Laminin.
After talking to the Laminin manufacturer, he said that Laminin can work only with 'enzymatic (pepsin)' antigen retrieval method. So we changed the method, now the Laminin works well, but we don't get any signal from neither PAX7 nor eMHC.
Do you have any suggestions for us for optimizing this staining protocol?
thanks!
Interesting that the anti-"laminin" antibody only works with a pepsin digest. I found a few papers that stained for "laminin" and they all used heat-based antigen retrieval. I had used a specific anti-laminin antibody(gamma or LAMC1) and it worked with the heating method.
References for laminin stains:
"Heat induced antigen retrieval was carried out by heating tissues in Tris saline buffer using microwave oven - two cycles each at 96°C for 6 minutes." (PMID: 25364175)
"laminin (Thermo Fisher Scientific, polyclonal antibody, PA1-16730 clone, high-temperature antigen recovery using 0.01 M citrate recovery solution pH 6 for 15 minutes, 1 : 50 dilution for 60 minutes" (PMID: 30298138)...
You may have to switch to another laminin antibody or to frozen tissue. That also seems to depend to some extent on your eMHC antibody? Do you do double immunofluorescence stains? Even if you have antibodies made in the same species, you still can use them for a double stain by doing aa sequential stain. If the laminin does not work well with the heat antigen retrieval, you can use a more sensitive detection system such as polymers (e.g., https://www.leicabiosystems.com/ihc-ish-fish/novocastra-antibodies/detection-systems-buffers/products/novolink-polymer/; https://vectorlabs.com/immpress-hrp-horse-anti-mouse-igg-peroxidase-polymer-detection-kit.html; https://www.cellsignal.com/products/secondary-antibodies/boost-ihc-detection-reagent-hrp-rabbit/8114?N=4294960088+102287+4294964832+4294956287&fromPage=plp)
Good luck.
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