formalin fixed- pll coated- primary followed by secondary then DAPI- permeabilization was done with triton X
Almost looks as though you have switched your colours around and the green channel is detecting your DAPI. The microscope will give you the image based on the camera settings you have provided. So if you have assigned the colour green to your 350 channel and blue to your 488 channel, then your image will reflect that. I would suggest going back to your image properties and checking that everything is set-up properly. You can always re-assign the colours so DAPI is blue if you need to.
Thank you Ashleigh. Our filter settings were DAPI- A4 and Green - GFP , will cross check the image properties. We had also processed another sample along with this one which showed blue nuclei staining and green extracellular staining, which got us wondering whether this particular sample was the issue. Could a processing step for IHC or prolonged decalcification of the specimen or permeabilisation with Triton X 100( did not perform the antigen retrieval step with pepsin prior to primary antibody incubation ) give extracellular staining ?