Slowfade gold antifade mounting reagent with 49 6 diamidino 2 phenylindole dapi

Sixteen-micrometer frozen sections were collected, air-dried briefly, rehydrated in PBS, blocked with protein block (5% donkey serum in PBS containing 0.1% Triton X-100), and incubated over night at 4 °C with primary antibodies including: chicken anti-GFP 1:400 (A10262, Life Technologies, Carlsbad, CA, USA) and anti-CD11c. After PBS washes for three times, the slides were incubated with corresponding Alexa Fluor 488 and Alexa Fluor 594 conjugated secondary antibodies, 1:400, (Jackson ImmunoResearch Laboratories, West Grove, PA) and counterstained for nuclei with SlowFade Gold Antifade mounting reagent with 49,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Immunofluorescence microscope images were collected using an FVII digital camera with Extended Focal Imaging (EFI) function (Olympus America, Center Valley, PA). The camera acquisition time for EGFP fluorescence was optimized and set a priori for each tissue.