To me this looks like blood cells. The reason why you are getting an overlap with DAPI could be just leaking of the green channel to the blue channel in the microscope (I think you are not using confocal, right?)

The sample preparation seems quite bad, there seems to be overlapped slices of tissue and uneven surface (the blurred areas). If the general quality is that bad, perhaps that's the reason for so many blood cells present. Can you get another sample?

Also, 1:400 could be a bit too concentrated... You know what? Try it without any antibodies, just DAPI. Do everything the same way you are doing until the antibody step, then skip both antibodies (no incubation at all) and go directly to mounting and imaging. If you will still get the signal, then it's blood cells for sure.

Autofluorescence is a phenomenon where materials including

tissues and cells emit background fluorescence in the absence of any

experimental fluorophore, leading to degradation of signal-to-noise ratio. Sources of autofluorescence include NADH/NADPH, flavins and flavoproteins, the extracellular matrix proteins elastin and collagen, and lipofuscin, an insoluble lysosomal polymer that accumulates in aging

tissues. The level of autofluorescence can vary by cell/tissue type and with choice of fixative. Autofluorescence tends to be strongest in shorter

wavelength channels (green/yellow/orange emission). Although there are reported approaches to reduce autofluorescence in tissue such as

short wavelength irradiation or quenching with sodium borohydride or pigments, these will also affect experimental fluorophores and target

antigens in the sample as well. If you are concerned

your sample may be autofluorescent, particularly for a dim signal, you can perform a control to compare intensities with and without antibody/fluorophore.

Thank you Mohamed Elkablawy . Seem to have found the culprit. Our tissues are paraffin embedded and I have found in some papers online that this actually leads to autofluorescence. I went back and looked at a slide without any antibody or IHC treatment and saw very similar fluorescence across both green and DAPI field.

Thank you Yulia Panina for your help. This was our first trial treating slides and these samples had trouble staying on the slides. Since that I've found different techniques that help keep the samples in better shape which have worked well.