Immunofluorescence Staining of CD68 in Tissues

The slides were rinsed with PBS and masked in a solution that contains 10% goat serum at ambient temperature, followed by an incubation period at 4°C with rat anti-CD68 antibody (1 : 250, Serotec, Oxford, UK). Finally, the sections were treated with Alexa Fluor 555-donkey anti-rat IgG for 45 minutes at 37°C (1 : 1000, Invitrogen, Carlsbad, USA). 4′,6-diamidino-2-phenylindole (DAPI; 1 : 500, Invitrogen) was used to counterstain the nuclei for 30 seconds. A confocal microscope was used to capture the fluorescent pictures (Leica SP5, Germany). The intensity of immunofluorescence staining was measured using the Image-Pro Plus 6.0 software (Media Cybernetics, USA) to calculate the integrated optical density (IOD) of positive expression from six randomly selected sections, as described previously [16 (link)].