Diazabicyclooctane dabco

Prelabeled brainstem-projecting cells and superficial layer interneurons were intracellularly injected with 0.3–0.5% Neurobiotin (Vector Laboratories) during patch-clamp recordings. Brain slices were fixed overnight in 4% formaldehyde and 14% picric acid in 0.1 M PB. Following a thorough rinse in PBS, the slices were incubated in streptavidin-Cy2 (1:1000, Jackson ImmunoResearch) in 0.3% Triton X-100 and 1% BSA in 0.1 M PB for two hours at room temperature. The slices were then rinsed in 0.01 M phosphate buffered saline (PBS) and mounted in glycerol containing 2.5% diazabicyclooctane (DABCO; Sigma). Labeled cells were analyzed by either confocal or conventional fluorescence microscopy.

Positive brains isolated and fixed with 4% PFA in 0.1M phosphate buffer, then cryoprotected with 15% and 30% sucrose in phosphate buffer. Brains were embedded and frozen in OCT (TissueTEK) and sectioned 20 μm on slides (Superfrost glass slides, Thermo Scientific) by cryostat (Leica). Sections were dried at room temperature (RT) before antigen retrieval was performed with 1X Citrate buffer, at 80 °C for 15 minutes. Sections were blocked with 5% Normal donkey serum with 0.01% Triton X-100 and 0.1M phosphate buffer for 2h at RT. Sections were incubated overnight at 4 °C with primary antibody solutions made with blocking buffer. Sections were washed with phosphate buffer at RT and incubated with secondary antibody solutions with blocking buffer for 3h at RT. Sections were washed again as above and incubated with 1:1000 DAPI to stain the nuclei. The sections were rinsed once with phosphate buffer and dried at RT. Sections were mounted in mounting media containing diazabicyclo-octane (DABCO; Sigma) as an anti-fading agent and visualized using Zeiss Apotome 2 microscope.