Alexa 568 anti mouse igg

For myosin staining, T cells migrating in collagen gel were fixed with 4% paraformaldehyde for 20 min at RT and then permeabilized with 0.5% Triton-X 100 (Sigma Aldrich T9284) for 15 min. For actin and tubulin staining, cells migrating in collagen gel were washed three times with PHEM solution (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM acetate) and then fixed with 4% paraformaldehyde + 0.2% glutaraldehyde + 0.1% Triton-X-100 in PHEM buffer for 15 min at RT. Cells were then incubated in blocking solution (1% BSA in DPBS) for 1 h. Primary antibodies (anti phospho-Ser-19 myosin Light chain 2, Cell Signaling 3671; or anti-tubulin, Sigma T9026, clone DM1A) were added o.n. in blocking solution at 4 °C (1:50 dilution). The following day, samples were incubated with secondary antibody (Alexa-488 anti-rabbit IgG, Invitrogen A32731; or Alexa568 anti-mouse IgG, Invitrogen A10037) in presence of phalloidin-Alexa647 (Invitrogen A22287, to stain actin) for 1 h at RT in blocking solution. Finally, samples were washed, stained with DAPI, and mounted using Vectashield Mounting Medium (VectorLabs H-1900-10). Images were acquired using a spinning-disk (Yokogawa CSU-W1 T1) Ixplore IX83 microscope (Olympus) equipped with ORCA-Flash 4.0 V3 (sCMOS Hamamatsu) camera and CellSens Dimension software (Olympus, v4.1.1).

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.

Cells grown on coverslips (18 × 18 or 22 × 22 mm²) were briefly washed twice using 1X Phosphate Buffered Saline (PBS) and fixed for 10 min in 4% Paraformaldehyde (Sigma, P6148) in PBS, pH 7.4 at RT, washed thrice in 1X PBS (5 min each), followed by permeabilization in 0.5% Triton-X-100 (in PBS) for 10 min at RT. Cells were blocked in 1% Bovine serum albumin (BSA) (Sigma, A2153) in 1X PBS, for 30 min, washed thrice with 1X PBS and incubated in primary antibodies for 90 min at RT and secondary antibodies for 60 min at RT, with washes in between using 1X PBS. Primary antibody was diluted in 0.5% BSA (prepared in 1X PBS): anti-Nucleolin (ab13541), 1:300, anti-H2B (Millipore, 07-371), 1:100. Following secondary antibodies were used after diluting in 1X PBS +0.1%Triton X-100 (PBST): anti-mouse IgG-Alexa 568 (Molecular Probes), 1:1000; anti-rabbit IgG Alexa 488 (Molecular Probes) after which cells were washed thrice in 1X PBST. Cells were mounted in Slowfade Gold Antifade (Invitrogen, S36937). Cells were imaged on a Zeiss LSM710 confocal microscope with 405 nm, 458 nm and 561 nm laser lines at 2% power using a 63X oil immersion objective, NA 1.4 at 2.5X digital zoom. X-Y resolution: 512 pixels X 512 pixels (1 pixel = 0.11 µm). Confocal z-stacks were collected at an interval of 0.32 µm.