Rabbit anti c fos

Immunohistochemistry was performed as previously described (Lin et al., 2018 (link)). Mice were transcardially perfused with 4% PFA in PBS and the spinal cord were stored in 1% PFA at 4°C overnight. Samples were transferred to 20% and 30% sucrose solution for dehydration at 4°C and embedded with O.C.T medium (Fisher Healthcare). Frozen blocks were cut with cryostat (Thermofisher, USA) or vibratome (Leica, VT1000 S) to prepare 20-60 μm-thick transverse slices. For somatodendritic analysis of ErbB4+ cells, 400-μm thick slices were prepared. Slices were washed with PBS and blocked with PBS containing 0.5% Triton-100 and 10% donkey serum for 1 hr at RT, before overnight incubation of primary antibodies at 4°C. After wash, slices were incubated in AlexaFluor secondary antibodies (Jackson ImmunoResearch) for 2 hr at RT. Slices were washed, mounted onto slides and covered with SlowFade Diamond Antifade Mountant (catalog #S36972; Thermo Fisher). The following primary antibodies were used for immunostaining: mouse anti-c-Fos (#271243, 1:500; Santa Cruz), rabbit anti-c-Fos (#ABE457, Millipore), chicken anti-GFP (#1020, 1:1,000, Aves), anti-RFP (#600-401-379-RTU, 1:500, Rockland), and rabbit anti-NeuN (#R-3770-100, 1:500, Biosensis). Alexa-647 conjugated streptavidin (#S21374, 1:200, Life technologies) was used for visualization of biocytin.

For double labeling of nicotine-induced c-Fos and GAD67-GFP containing GABAergic neurons, tissues were washed and incubated with a PBS cocktail consisting of 0.3% Triton X-100 and rabbit anti-c-Fos (1:1000, Millipore Corporation, Temecula, CA) at 4°C for 48–72 h. The sections were then incubated in Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:100; Jackson ImmunoResearch Laboratories, Inc.) in 0.1 M PBS for 2½ h. After washing in PBS, sections were rinsed in PBS, mounted and cover-slipped using Vecta Shield (Vector Laboratories Inc., CA) anti-fade mounting media.
Controls for each experiment were performed to determine whether the primary or the secondary antibodies produced false-positive results. The controls involved omission of the primary and/or secondary antisera to eliminate the corresponding specific labeling. Nonspecific activation of c-Fos was assessed by evaluating the CNS expression of c-Fos in animals receiving i.p. injection of normal Physiological Saline (PS).

Ninety minutes after the CFC test, mice were deeply anesthetized with ketamine/xylazine (150/15 mg/kg) and transcardially perfused with 1× phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in 1× PBS. Brains were extracted and post-fixed overnight at 4°C in 4% PFA and then transferred to a 30% sucrose in 1× PBS at 4°C for two days. Thirty-five μm coronal sections were collected on a cryostat and stored in cryoprotectant at −20°C.
For immunohistochemistry, sections were washed in 1× PBS and blocked at room temperature (RT) for 2 h in 10% normal donkey serum (NDS) in 1× PBS with 0.5% Triton-X (PBS-T). Sections were incubated with primary antibodies (1:2,000 rabbit anti-Arc (Synaptic Systems); 1:500 chicken anti-GFP (Abcam); 1:1000 rabbit anti-c-Fos (Millipore)) diluted in 5% NDS in 1× PBS-T overnight at 4°C. Sections were rinsed in 1× PBS-T and incubated in secondary antibodies (Jackson ImmunoResearch; 1:500 donkey anti-rabbit Cy3; 1:500 biotinylated donkey anti-chicken) in 1× PBS-T for 2 h at RT. Sections were rinsed in 1× PBS-T and incubated in tertiary antibody (1:250 avidin Cy-2 (Jackson ImmunoResearch)) and 1:1000 DAPI for 1 h at RT. Sections were washed in 1× PBS, mounted onto slides, and coverslipped with ProLong Gold (Invitrogen). See the Life Sciences Reporting Summary for additional information.