Rabbit anti cfos

Vglut2-IRES-Cre mice were injected with AAV-retro-DIO-GFPL10 into the POA and AAV-retro-DIO-mCherry into the DMH. Four weeks following the injection, mice were exposed to a warm (38^oC) or cold (10^oC) environment in an incubator for two hours, then were perfused with PBS and 4% PFA and processed following the immunohistochemistry protocol described above. POA-projecting LPB^Vglut2 neurons were labeled with GFP. DMH-projecting LPB^Vglut2 neurons were labeled with mCherry. Warm- or cold-activated cFos in the LPB neurons were labeled by cFos immunostaining (rabbit anti-cFos, Synaptic systems, #226003, 1:10000) colored with Fluor 647-conjugated secondary antibody (Alexa Fluor 647 conjugated goat anti-rabbit, Invitrogen, #A21244, 1:1000). Images were captured on a Nikon A1R confocal microscope.

Mice were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Brains were then placed in a 30% sucrose solution for 2 days. The brains were frozen in optimal cutting temperature embedding medium, and 16-μm (for FISH) or 40-μm [for immunofluorescence (IF)] coronal sections were cut with a vibratome (Leica, no. CM3050 S). For FISH experiments, the slices were mounted on SuperFrost Plus slides and air-dried. The multicolor FISH experiments were performed following the instructions of the RNAscope Fluorescent Multiplex Assay (ACD Bioscience). For IF, cryostat sections were collected and incubated overnight with blocking solution (1× PBS containing 5% goat serum, 2.5% bovine serum albumin, and 0.1% Triton X-100), then treated with the following primary antibodies, and diluted with blocking solution for 1 day at 4°C: rabbit anti–c-Fos (1:2000; Synaptic Systems, no. 226003), chicken anti-GFP (1:2000; Aves Labs, no. GFP-1010), and chicken anti-mCherry (1:2000; Novus Biologicals, no. NBP2-25158). Samples were then washed three times with washing buffer (1× PBS containing 0.1% Triton X-100) and incubated with the Alexa Fluor–conjugated secondary antibodies for 2 hours at room temperature. The sections were mounted and imaged using a Zeiss LSM800 confocal microscope or the Olympus VS120 Slide Scanning System.

After all the experiments, mice were perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. The brains were extracted, postfixed overnight in 4% PFA at 4 °C, and cryoprotected in 30% sucrose. Brains were sectioned to a thickness of 30 mm using a sliding freezing microtome (Leica SM2010R) and preserved in a cryoprotectant 30% ethylene glycol, in PBS). Free-floating sections were washed in PBS, incubated for 1 hr in blocking solution (10% normal goat serum (NGS) and 0.3% Triton X-100 in PBS), and incubated overnight at 4 °C with primary antibodies (rabbit anti-β2-AR, Thermo Scientific, Alomone Labs, 1:500; Mouse anti-CaMKIIα, 1:500; rabbit anti c-fos, Synaptic Systems, 1:500) in 0.1% Triton and 3% NGS in PBS. Sections were then washed with PBS 4 times and incubated for 2.5 h at room temperature with secondary antibodies (goal anti-rabbit, Alexa Fluor 594, 1:300; goal anti-mouse, Alexa Fluor 488, 1:300; goal anti-mouse, Alexa Fluor 594, 1:500; goal anti-rabbit, Alexa Fluor 647, 1:300; goal anti-rabbit, Alexa Fluor 405, 1:300) in PBS. Finally, sections were washed in PBS 4 times, mounted on slides and sealed with mounting medium (Fluoromount-G, eBioscience, San-Diego, CA, USA). Mounted slides were imaged using an inverted laser scanning confocal microscope (LSM 880; Carl Zeiss, Oberkochen, Germany).

Mice were deeply anesthetized with an 3% isoflurane and perfused with 20 ml ice-cold phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA). Brains were carefully removed and postfixed in 4% PFA for 6–8 h, and cryoprotected with 30% sucrose for 48 h. The brain was 30 μm thick sections were coronally prepared using a freezing microtome (Leica CM1950). For immunofluorescent staining, the sections were incubated with PBS containing 0.3% Triton X-100 for 1 h at room temperature and subsequently allowed to react with primary antibodies (rabbit anti-glutamate, 1:500, Sigma; rabbit anti-c-Fos, 1:1000, Synaptic Systems) at 4˚C overnight. After washing with PBS, the sections were subsequently coupled with the corresponding fluorophore-conjugated secondary antibodies for 1.5 h at room temperature. Finally, after rinsing 3 times in PBS, sections were mounted with DAPI staining. Confocal images were acquired under a 10x or 20x objective with a 1024 × 1024 resolution using the Olympus confocal microscopes (FV3000, Olympus). Double fluorescence labelled cells were manually counted using ImageJ software. The Paxinos and Franklin atlas (2013) was employed to define the regions of interest.

Sections were washed with phosphate-buffer saline 0.10 M triton X-100 (Sigma-Aldrich, Madrid, Spain; Cat# T9284) (1%) (PBST) and then incubated in a blocking solution (1.50% Donkey serum; Cat# D9663; Sigma-Aldrich, Madrid, Spain) in PBST for 30 min at room temperature. Subsequently, they were incubated with a polyclonal primary antibody, rabbit anti-cFos (1:500; Cat# 226003; Synaptic Systems, Göettingen, Germany) in PBST with Donkey serum (1.50%), in smooth agitation at 4°C for 24 h. After washing with PBST, sections were incubated with donkey anti-rabbit Alexa Fluor 555 conjugate (1:250; Cat # A-31572; Thermo Fisher Scientific, Madrid, Spain) in PBST with Donkey serum (1.50%) for 2 h at room temperature. Then, sections were rinsed with PBST and mounted with FluorSave reagent (Cat# 345789; Calbiochem; Millipore; Merck KGaA, Darmstadt, Germany).