Alexa 568 anti rabbit igg

The tibialis anterior (TA) muscle was dissected from perfused mice, transferred into a 30 % sucrose solution for 2 days, and cut using a cryostat at 14-μm thickness. To visualize muscle fiber size and location of nuclei, the sections were stained by first blocking for 1 h at room temperature (0.1 % Triton X-100, 3 % BSA, 5 % goat serum in PBS) and then incubated with an antibody against laminin (L9393, Sigma; 1:100) for 24 h in blocking solution. The sections were washed three times in PBS and incubated for 3 h with secondary antibodies (Alexa-568 anti-rabbit IgG, Life Technologies; 1:1000). After washing with PBS, sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Sigma; 1:1000), washed with PBS, and mounted using Vectashield. The area outlined by laminin was measured using ImageJ software. At least 300 muscle fibers per mouse were randomly selected and used for this analysis. Myonuclei located in the center of muscle fibers were counted. At least 1000 nuclei per mouse were counted.

Mice were anesthetized with isoflurane and transcardially perfused with preperfusion solution (9 g NaCl, 5 g sodium nitrate, 1,000 u heparin in 1 L distilled water). Brains were incubated in 4% paraformaldehyde overnight at cold room and sectioned with a vibratome in 50 μm on the following day. For immunofluorescence staining, sections were incubated in 10 mM sodium citrate solution (pH 9.0) for 30 min at 80 °C in a water bath. Following the antigen retrieval, the sections were blocked in 0.1 M PBS buffer containing 0.1% triton X-100, 5% normal goat serum or normal donkey serum, and 5% bovine serum albumin for 2 h at room temperature and then incubated with anti-TRPV1 (1:1,000, Alomone Labs, cat # ACC-030), anti-c-fos (1:500; Abcam, cat # ab7963), anti-GFP (1:1,000, Abcam, cat # ab13970), anti-mCherry (1:1,000, Novus Biologicals, cat # NBP2-25157), and anti-POMC (1:1,000, Phoenix, cat # H-029-30 [35 (link)]) antibodies for 72 h at cold room. And then sections were washed 3 times in PBS and incubated with Alexa 568 anti-rabbit IgG (1:500; Life Technologies, cat # A10042), Alexa 568 anti-mouse IgG (1:500; Life Technologies, cat # A11004), or Alexa 488 anti-chicken IgG (1:500, Abcam, cat # ab150169) for 2 h at room temperature. Tissues were washed, dried, and mounted with VECTASHIELD media containing DAPI. Images were acquired using a Leica SP2 confocal microscope.

Y-27632 and 4’,6-diamidino-2-phenylindol (DAPI) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Polysciences (Warrington, PA), respectively. Matrigel (growth factor-reduced) was purchased from Corning Incorporated (Corning, NY). Rabbit polyclonal antibodies against human Solo were prepared as previously described [14 (link)]. Other antibodies were purchased as follows: anti-FLAG (M2; Sigma-Aldrich, St. Louis, MO), anti-GFP (A6455; Life Technologies, Camarillo, CA), anti-β-actin (AC-15; Sigma-Aldrich), anti-K18 (DA-7; BioLegend, San Diego, CA), anti-GM130 (CSB-PA600856ESR1HU; Flarebio Biotech LLC, College Park, MD), anti-β4 (58XB4; BioLegend) for immunofluorescence, and anti-β4 (Clone 7; BD Biosciences, Franklin Lakes, NJ) for immunoblotting. Secondary antibodies, anti-mouse IgG-Alexa 488, anti-mouse IgG-Alexa 568, anti-rabbit IgG-Alexa 568, and anti-rat IgG-FITC, were purchased from Thermo Fisher Scientific (Waltham, MA).

Indirect immunofluorescence was performed as described previously. Anti-Cx26 mAb clone CX-12H10 (Thermo Fisher Scientific) and anti-p120 Catenin pAb (Sigma-Aldrich, St. Louis, MO, USA), anti-GM130 pAb (Sigma-Aldrich) were diluted at 1:150, 1:200, and 1:3500, respectively. After fixation with acetone, cells are incubated with the diluted primary antibodies. Specific signals were revealed by anti-mouse IgG-Alexa 488 (Thermo Fisher Scientific) and anti-rabbit IgG-Alexa 568 (Thermo Fisher Scientific). Nuclei were stained with DAPI (KPL, Gaithersburg, MD, USA) at a concentration of 0.5 µg/mL.