Alex488

Mice were anesthetized with choral hydrate and perfused with saline followed by 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS). The brains were removed, fixed in 4% paraformaldehyde overnight, and subjected to dehydration in increasing saccharin solutions (20–30%) at 4^ºC. The frozen coronal slices of 20 μm thicknesses were prepared and stored at -20^ºC in 20% ethanediol PBS solution containing 20% saccharin. Brain sections were incubated in 3% normal goat serum and 0.2% Triton-X for 1h. Then they were incubated in rabbit anti-Parvalbumin (Swant, Bellinzona) or mouse anti–corticotropin-releasing factor (CRF; Abcam) antibody overnight at 4ºC. Slices were rinsed in PBS then incubated in donkey anti-rabbit Cy3, Alex488, DyLight 647, or donkey anti-mouse Cy3 (Jackson Immunoresearch) for 1h and 4',6-diamidino-2-phenylindole (DAPI) for 10min, then mounted after rinsing. Images were acquired on a microscope using a 20× air objective, a 40× objective (IX-83; Olympus) or a 63× oil immersion objective (Zeiss 510; Carl Zeiss Jena).

Cells were seeded on glass coverslips and fixed with 4% PFA in PBS for 15 min and subsequently permeabilized and blocked for 20 min in PBS containing 0.1% Triton X-100 and 5% FBS. Detection was performed by incubating with primary antibodies at 1:500 dilutions in PBS and secondaries (Jackson laboratories, Alexa633 coupled donkey anti-goat and Alex488 coupled anti-rabbit) at 1:2000 for 1 h in PBS/0.1% Triton. Four 1-min rinses in PBS were performed after each incubation. Cells were then mounted in Fluorescent Mounting Medium (DakoCytomation) supplemented with 1 µM Hoechst 33,342. Microscopy was performed using a 63 X Oil Immersion lens on a Zeiss Elyra microscope using the Zeiss Zen software. Images were acquired in 2 or 3 channels using sequential excitation/emission. In images shown only linear changes in intensity and contrast were performed (K = 0).