C fos antibody

After overnight fasting, mice were injected with either saline (same volume than glucose) or glucose (2g/kg, ip) and after 1 h from the injection they were perfused. In another set of experiments, 2h fasted mice were injected with 2DG (200 mg/kg, ip) and after 45 minutes from the injection they were transcardially perfused. Brain sections were then immunostained for cfos (rabbit anti-c-fos antibody; Santacruz, 1:2000). No staining was performed to visualize POMC neurons since mice were expressing in this neuronal population tdTomato which is per se fluorescent. Sections were mounted on glass slide with vectashield (Vector lab) and analyzed with fluorescent microscope.

Prior to experimentation, males were isolated from female scent in a room separate from other mice for at least 2 days. On the day of the experiment, male mice were singly housed in a clean cage in a quiet room for three hours prior to female exposure. The male mice were then exposed (at 6:00 PM under dim red light) to urine (60 μL) collected from hormonally primed estrus stimulator female mice. The urine was placed just out of reach of the mice to prevent direct contact. After 90 minutes, males were killed, and brains were collected in fixative. Sections were stained with c-Fos antibody (Santa Cruz Biotechnology; catalog number sc-52 RRID:AB_10160513; 1:1000). Neuroanatomical landmarks were used to identify the counting region as depicted in the corresponding figure. Quantification was performed on biological replicates consisting of c-Fos nuclei within the defined region from a minimum of three unilateral sections. C-Fos-positive cells were quantified by an experimenter blinded to the treatment group. While the c-fos IHC produced a range of staining intensity, only those cells that were darkly stained were included in the quantification. The numbers from each biological replicate were then averaged across all the animals in that group. Slides were coded to blind the researcher to treatment group during analysis.

Mice were perfused transcardially using 30 ml phosphate buffered saline (PBS) followed by 30 ml 4% paraformaldehyde in PBS. Brains were dissected out and postfixed for 24 hours in 4% paraformaldehyde at 4°C, t hen transferred to 30% sucrose until fully saturated. Slices of the BNST (45 μm) were prepared on a Leica 1200S vibratome and stored in 50/50 glycerol/PBS at −20°C for FOS i mmunohistochemistry. Slices were first washed in PBS, then incubated in 50% methanol and followed by 3% H₂O₂. Slices were washed in PBS again followed by a 24 hour incubation at 4°C in 0.5% BSA, 0.3% Triton-X100, and c-fos antibody (1:3000; Santa-Cruz). The next day, slices were washed in Tris-HCl, NaCl, Tween (TNT) buffer, incubated in Tris, NaCl blocking (TNB) buffer, and then incubated in goat anti-rabbit HRP-linked IgG (1:200) in TNB buffer for 30 minutes. Slices were washed in TNT, and tyramine signal amplification (TSA) with Cy3 was performed using a kit (Perkin Elmer).
Slices were mounted with Vectashield on glass slices and imaged using a Zeiss AXIO Zoom V16 microscope with ZEN pro 2012 software. FOS-positive cells from at least 3 sections per animal were counted using NIH image software separately in the dorsolateral BNST (dlBNST), dorsomedial BNST (dmBNST) and vBNST.