I'm planning on doing a combined staining on FFPE TMA slides: ISH (red dots) and a nuclear IHC in DAB (brown nuclei). We would like to also visualize the collagen in this same staining. However, with a Masson trichrome, a lot of the tissue will be stained red, which would render the ISH dots invisible. Is it possible to use just the Light Green (with Weigerts Haematoxylin) to stain the collagen?
I tried it and it works. It sadly doesn't look great combined with the ISH and IHC.
You can also just switch to FISH (Floresent ISH) and combine it with Collagen antibody than you can do the following:
Nuclei - (DAPI) - 350 nm Blue channel
FISH 488 nn probe - Green channel
Collagen 545 or 595 nm secondary - Red channel. This way you can easilly resolve your target and it's relevance to collagen. You will need laser confocal microscope to produce such pictures as normal florecent microscope will probably make difficult to resolve the samples, highly depend on the signal streight though.
We are going to do that for the ISH probe, but when we add the IHC antibody (p63), we can't do this combination anymore because they're both anti-mouse antibodies. That's why we were thinking of using the trichrome as an alternative.
In that case I recommand to work with FAB-secondary antibody.
Use for the first mouse primary AB the Anti-mouse-FAB-Flu AB and for the second primary AB Anti-mous HL-Flu AB for detection. Blocking step with high concentrated normal serum (min. 20%) will also help to prevent unspecific background staining. To run a negativ control is highly recommanded.
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