Macrophages ( Microglia) specifically express GFP, in this transgenic mouse. Note that rbcs are positive for Alexa 488; this is due to formalin-induced fluorescence, particularly strong in green wavelength but also in the visible red wavelength

HIER is best but Proteinase K is also good, for antigen retrieval. Anti NeuN ab is directly conjugated to Alexa Fluor 647. Chicken anti GFP was detected using Goat anti chicken-biotinylated ( Abcam) and then stAvidin-488 Invitrogen.