Combine brightfield and fluorescence


our slide scanner has troubles detecting small tissue biopsies using the darkfield preview.
Hence, I would like to combine a brightfield counterstain (tissue detection) with FITC IF imaging (specific antibody) on the same slide.
Do you have any idea if this would be possible?

Best regards,

Hello Mario,

Could you identify which slide scanner you are using?

Adding a brightfield counterstain may affect your IF image, as some counterstains are also fluorescent (eosin as an example of a counterstain with green fluorescence).

Dear George,

we are using a 3DHistech P250 Flash II with DAPI, FITC, TRITC and Cy5 Bandpass filters.
I’ve read that eosin causes problems in IF and hematoxylin might interfere with DAPI. Hence my question.

Best regards,

I don’t have a recommendation for a brightfield stain that will not affect fluorescence, but there might be an alternative approach.

The manual for your scanner does describe a detection approach method similar to one that I use with fluorescent samples that have low brightfield contrast. The approach is to draw an outline around the tissue with a dark marker (black Sharpie) and then focus + scan within that marker outline. For tissues that are hard to see by eye, I shine a small flashlight onto the edge of the slide while drawing the outline to visualize the tissue. Has that method been tried?

Hi George,

thank you for your suggestion. I am aware of the scanners ability to scan “inside marker area”. But since this type of staining will be done very frequently, I would prefer to find a solution not requiring manual intervention.
I will try to apply an autofluorescence quenching solution on the slide, which “stains” the tissue darker and should increase darkfield tissue detection without interfering with IF markers.

Best regards,