Mouse anti cgrp

As reported before [32 (link)], the mice were anesthetized with 1% pentobarbital sodium and then perfused with normal saline followed by a solution of 4% paraformaldehyde (PFA). The DRGs were post-fixed in 4% PFA for 4 h and then dehydrated sequentially in 20% and 30% sugar solutions. Each dehydrated DRG sample was excised into 10-µm-thick serial sections, which were then mounted on glass slides. After antigen retrieval and blocking with 5% fetal bovine serum, the DRG sections were co-incubated overnight at 4 °C with rabbit anti-TET2 antibody and DAPI with one of the following antibodies: mouse anti-NeuN (1:500, Sigma), mouse anti-NF200 (1:500, Sigma), or mouse anti-CGRP (1:500, Abcam). Subsequently, after washing the slides three times with phosphate buffered saline (PBS) (for 5 min each time), the sections were incubated with a mixture of Alexa Fluor 488 goat anti-rabbit IgG (H + L) and Alexa Fluor 568 goat anti-mouse IgG (H + L) (1:200, Invitrogen, Life technologies™, USA) 1 h in dark condition. Images were captured under a fluorescence microscope. We acquired at least 9 locations in the DRG per mouse: we quantified 3 images per section and 3 sections per mouse. We quantified at least 3 mice per experiment blindly.

Animals were perfused with ice-cold 4% PFA in PBS. Brains were removed, post-fixed overnight at 4 °C in 4% PFA, and rinsed 3 times in PBS. Prior to sectioning, brains were embedded in a BSA-gelatin mixture consisting of 0.4% gelatin, 23.3% BSA, 5.9% formalin, and 0.32% glutaraldehyde. Brains were then sectioned at 40–50 µm on a Leica vibratome. Sections were blocked and permeabilized in 10% normal donkey serum (NDS) with 0.4% Triton X-100 in PBS for 1–3 hr. Antibodies were diluted in a buffer consisting of 5% NDS and 0.4% Triton X-100 in PBS and incubated for 1–2 nights at room temperature. The following primary antibody concentrations were used: Mouse anti-CGRP (Abcam, 1:500–1:1,000); Rabbit anti-NPY (Cell Signaling, 1:500); Goat anti-ChAT (Millipore Sigma, 1:500); Rabbit anti-Ucn (Sigma-Aldrich, 1:500); Chicken anti-TH (Abcam, 1:1,000); Chicken anti-GFP (Aves, 1:2,000). Sections were rinsed 3 times with PBS, then incubated in secondary antibodies for 1.5–3 hr at room temperature. All secondary antibodies were used at 1:1,000 in 5% NDS and 0.4% Triton X-100 in PBS (Key Resources Table). Finally, sections were incubated in DAPI (Invitrogen, 1:10,000) for 10 minutes at room temperature, rinsed twice with PBS, and mounted with Vectashield hard-set medium (Vector Labs). Samples were imaged on either an Olympus VS120 fluorescent microscope or a Zeiss LSM 800 confocal microscope.

Mice were anesthetized and perfused with 4% formaldehyde in 0.1m phosphate‐buffered saline (PBS, pH 7.4). DRGs were dissected rapidly, postfixed in the same fixative solution at 4 °C for 4 h, and cryoprotected in 30% sucrose overnight. A series of 20‐µm transverse sections were cut on a cryostat. The sections were blocked with 5% goat serum and 0.3% TritonX‐100 in 0.01 m PBS at room temperature for 1 h and then incubated with rabbit anti‐Pou4f3 (1:800, GeneTex) plus biotinylated IB4 (1:200, Sigma), mouse anti‐CGRP (1:200, Abcam), mouse anti‐NF200 (1:200, Sigma), mouse anti‐glutamine synthetase (1:1,000, Sigma) or chicken β‐tubulin III (1:500, EMD Millipore) or with rabbit anti‐RALY (1:2,500, Abcam) plus mouse anti‐glutamine synthetase (1:1,000, Sigma) or chicken β‐tubulin III (1:500, EMD Millipore) at 4 °C over‐two nights. The sections were then incubated with species‐appropriate Cy2‐ or Cy3‐conjugated secondary antibody (1:500, Jackson ImmunoResearch), or FITC‐labeled Avidin D (1:200, Sigma) at room temperature for 2 h. Control experiments were performed in parallel. The images were captured with a Leica DMI4000 fluorescence microscope (Leica).

Animals were transcardially perfused with saline and 4% PFA. Brains were post-fixed overnight in 4% PFA at 4 °C, followed by immersed in 30% sucrose solution. Coronal sections (40 μm) were cut by a CM1950 Microtome (Leica). Immunostaining was performed as previously described (Zhang et al., 2018 (link)). The brain slices were permeabilized in 0.5% Triton X-100 in Tris-buffered saline, blocked with 100 mM glycine and 5% bovine serum albumin (BSA) containing 5% normal donkey serum. Tissue sections were subsequently incubated with diluted primary and secondary antibodies as indicated, nuclei stained with 6-diamidino2-phenylindole (DAPI), and slides mounted with antifade reagents. The primary antibodies used were: Guinea pig anti-c-fos (Synaptic Systems, Cat# 226004), Rabbit anti-Dsred (Takara, Cat# 632496), Goat anti-GFP (Abcam, Cat# 5450), Mouse anti-CGRP (Abcam, Cat# 81887). Slides were imaged with a confocal microscope (Olympus FluoView FV1200).

Mice were anesthetized with 10% chloral hydrate and perfused through the ascending aorta with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, the TGs were removed and post-fixed with 4% paraformaldehyde for 4 h. The samples were cut into 14-μm-thick frozen sections on a cryostat. The sections were incubated with primary antibodies (mouse anti-TRPV1, 1:1,000, Abcam; rabbit anti-TRPA1, 1:500, Abcam; rabbit anti-TRPM8, 1:500, Abcam; mouse anti-CGRP, 1:1,000, Abcam; mouse anti-NF200, 1:1,000, Abcam; mouse anti-IB4-FITC, 1:1,000, Sigma; rabbit anti-ZBTB20, 1:1,000, Atlas Antibodies AB; rat anti-ZBTB20, 1:2,000, Abcam) overnight at 4°C, followed by secondary antibodies (Alexa Fluor 555 donkey anti-rabbit IgG, 1:1,000 and Alexa Fluor 488-conjugated donkey anti-mouse IgG 1:1,000, Invitrogen) at room temperature for 2 h. The sections were then observed under an epifluorescence microscope. All images were made into figures using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA), with only minor adjustments to the contrast and brightness settings if necessary.