Hello everyone,
We are trying to perform laser microdissection on human muscle. We prepared our 15µm slides of muscle on PEN slides and performed IF and counterstained the slide with dapi (we need to capture the nuclei). The problem is that we have a huge autofluorescence with dapi (the other fluorescent antibody is ok) and we cannot see any nuclei labelled with dapi
Does one of you already succeeded to do dapi staining on tissue on PEN slide?
Any experience would be great....
Thank you very much
Fanny
Dear Fanny,
I have switched to using PET-membrane slides when working with fluorescence to avoid the problem with autofluorescence.
Kind regards,
Anna
Dear Fanny,
we've succeeded in the use of DAPI in PEN-membranes in different protocols.
With fresh-frozen mouse brain tissue, we performed fixation with Ethanol 100% and after washing, we just incubate with DAPI for 3 min. Then washed again and let the sample dry before visualization and cutting.
With paraffin-embeded mouse brain tissue, after classical deparaffination procedure, we stained with some stain of our interest, then rinse with Ethanol 75% and applied a drop of DAPI as counterstaining.
In both cases, we were able to visualize DAPI in the blue channel without problems.
Hope this helps!
Best
Teresa
Apart from PET membranes, we offer Membrane Slides with special, low autofluorescence materials (PPS, etc).
Please let me know if you require any additional information.
I use PET slides. For DAPI, I use Thermofisher DAPI Solution (62248, 1 mg/mL, 1:1000 dilution) when incubating with the secondary antibody. It works.
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