Fig. 3.

Double immunofluorescent staining of macrophages using two mouse monoclonal antibodies targeting CD68 and inducible nitric oxide synthase (iNOS) in formalin-fixed paraffin embedded colon from a normal rat. Images depict serial sections of normal colonic mucosa and submucosa. Staining for iNOS (unconjugated primary antibody, green), CD68 (biotinylated primary antibody, red), and DAPI (blue) is shown in the first, second, and third columns, respectively. Overlays of the green, red, and blue channels are shown in the fourth column. a–d Staining for CD68, iNOS, and DAPI reveals the presence of several macrophages (CD68-positive cells) lacking iNOS staining. e–h The iNOS single-stain control (tissue did not receive CD68 antibody) confirms the lack of iNOS staining observed in a–d. i–l The CD68 single-stain control (tissue did not receive iNOS antibody) shows CD68 staining consistent with that shown in a–d. m–p Negative control tissue (did not receive either of the primary antibodies) demonstrates some autofluorescence that can be distinguished from areas of true positivity (orange color in p). Scale bar = 100µm