Can get signal immunostain solution b

To evaluate HS and CS GAG chains, HT22 cells were stained with either monoclonal anti-HS antibody 10E4 (F58-10E4 clone, 1:250 dilution; AMS Biotechnology Ltd. Abingdon, UK, RRID:AB_10891554) or monoclonal anti-CS antibody CS-56 (anti-6S, 2S, 4S antibody) (1:100 dilution; Sigma-Aldrich Corporation, St. Louis, MO, USA, RRID:AB_476879). HT22 cells (2.0 × 10³/cm²) were grown on 24-well plate and treated with sodium chlorate. The cells were fixed with 4% paraformaldehyde for 15 min and then non-specific binding was blocked with 2.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Subsequently, HT22 cells were incubated in primary antibodies with 1% BSA or immunoenhancing reagent (Can Get Signal^® Immunostain Solution B; TOYOBO, Osaka, Japan, Cat# NKB-401) overnight at 4 °C. HT22 cells were washed with PBS and immersed in 2.5% BSA or Can Get Signal^® Immunostain Solution B (TOYOBO, Osaka, Japan) containing anti-IgM-FITC (fluorescein isothiocyanate, 1:250 dilution; Jackson ImmunoResearch, PA, USA) and then incubated with 10 µg/mL Hoechst 33258 (Molecular Probes, OR, USA) for nuclei staining. After washing with PBS, fluorescence images were captured with a fluorescence digital microscope (BZ-X800, Keyence, Osaka, Japan). The fluorescent intensity was quantified using Keyence image measurement and the analyzing software (BZ-X800 Analyzer, Keyence Corporation, Osaka, Japan).

Protocol was described previously (Harmansa et al., 2015 (link)). Each fly cross was set together with control and >10 wing imaginal discs from each genotype were processed in parallel. If the genotype could be distinguished, experimental and control samples were processed in the same tube. A representative wing disc was shown for all the experiments. Following primary antibodies were used; anti-Dpp (1:100; Matthew Gibson), anti-phospho-Smad1/5 (1:200; Cell Signaling, 9516S), anti-Brk (1:1000; Gines Morata), anti-Wg (1:120; DSHB, University of Iowa), anti-Ptc (1:40; DSHB, University of Iowa), anti-β-Galactosidase (1:1000; Promega Z378A), anti-mCherry (1:5000; Nigg lab, University of Basel). All the primary antibodies except anti-Dpp antibody were diluted in 5% normal goat serum (NGS) (Sigma) in PBT (0.03% Triton X-100/PBS). Anti-Dpp antibody was diluted in 5% NGS in Can Get Signal Immunostain Solution B (TOYOBO). All secondary antibodies from the AlexaFluor series were used at 1:500 dilutions. Wing discs were mounted in Vectashield (H-1000, Vector Laboratories). Images of wing discs were obtained using a Leica TCS SP5 confocal microscope (section thickness 1 μm).

Tissues fixed with formalin were sliced and stained with NFR and haematoxylin eosin (HE) solution. To identify BA and beige adipose cells, UCP-1 immunostaining was performed after activating the antigen with EDTA treatments (1 mM, pH 8.0, Kanto Chemical Co., Inc., Tokyo, Japan, Catalogue No. 14097-00) and heating at 95 °C for 20 minutes. Primary and secondary antibody reactions were performed using a rabbit anti-UCP-1 polyclonal antibody (Abcam Plc., Cambridge, UK, Catalogue No. ab10983) and Histstar^TM (Rb) for mouse tissue (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan, Catalogue No. 8470). The observations were made with microscopy (Olympus BX60, Olympus Corp.) and NIR fluorescence microscopy.
To determine endothelial cells, CD31 immunostaining was performed using a rabbit anti-CD31 polyclonal antibody (Abcam Plc., Catalogue No. ab124432) and an Alexa Fluor^® 488 goat anti-rabbit IgG second antibody (Thermo Fisher Scientific Inc., Waltham, MA, USA, Catalogue No. A11008), along with Can Get Signal immunostain Solution B (TOYOBO Co., Ltd., Osaka, Japan, Catalogue No. NKB-601). The stained tissues were observed with confocal microscopy (LSM 5 PASCAL, Carl Zeiss AG, Oberkochem, Germany) at an excitation wavelength of 488 nm and NIR fluorescence microscopy.