Alexa fluor 546 donkey anti rabbit igg

For immunofluorescent staining of SC35/SRSF2 and UCP1 in primary adipocytes, cells were fixed with cooled ethanol (95%) and glacial acetic acid (5%) (10 min, −20°C), permeabilized by Triton X‐100 (0.3%, 10 min), blocked with donkey serum (10% in PBST with Triton X‐100 (0.3%), 2 h), incubated with primary antibodies against SC35 (mouse anti‐SC35, Abcam ab11826, diluted 1:100 in PBST with Triton X‐100 (0.3%) and donkey serum (1%), overnight) or against UCP1 (rabbit anti‐UCP1, Abcam, diluted 1:200 in PBST with Triton X‐100 (0.3%) and donkey serum (1%), overnight) and fluorophore coupled anti‐mouse or anti‐rabbit secondary antibodies (Alexa Fluor 647 donkey anti‐mouse IgG, Life Technologies or Alexa Fluor 546 donkey anti‐rabbit IgG, Life technologies, both diluted 1:500 in PBST with donkey serum (1%). Slides were mounted with Vectashield Mounting Medium with DAPI for nuclear counterstain (Vector Labs), sealed with a coverslip and nail polish, and visualized with 60× oil immersion objective in a confocal laser‐scanning microscope (Olympus FluoView FV10i). Fluorescent signals were quantified with ImageJ software.

BODIPY 493/503 (#D3922) and DAPI (#D1306) were purchased from Invitrogen (CA, USA). SKL2001 was purchased from Selleckchem (Shanghai, China). Dexamethasone (#D4902), 3-isobutyl-1-methylxanthine (#I5879), insulin (#I2643), Oil red O (#O9755), MS-222 (#E10521), and Triton X-100 (#T8787) were purchased from Sigma-Aldrich (MO, USA). Antibodies against P42/44 (#9102), β-catenin (#9562), Phospho-β-catenin (#9561), PPAR-γ (#2443), Ubiquitin (#3936), GAPDH (#5174), HA-tag (#3724) as well as secondary HRP-conjugated antibodies against mouse (#7076) and rabbit (#7074) were purchased from CST (MA, USA) and used at 1:1,000 dilution for western blotting. Anti-FLAG-tag antibody (#F1804) and anti-FLAG-tag M2 affinity gel (#A2220) were from Sigma-Aldrich (MO, USA). Antibodies against Plin1 (#A16294), OSBPL2 (#A14199), α-Tubulin (#AC012), β-catenin (#A11512) as well as secondary HRP-conjugated Mouse Anti-Rabbit IgG Light Chain (#AS061) were from ABclonal (Wuhan, China). Antibodies against β-catenin (#BF0319) for zebrafish were from Affinity Biosciences (Changzhou, China). Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG were purchased from Life Technologies (CA, USA).

Cells were grown on coverslips in 24-well plates overnight. The samples were fixed in 10% formalin, and then were penetrated with 0.1% triton X-100 (Sigma). Then, the samples were blocked with goat serum (used at 1:10 dilution, #16210064, Gibico, USA) for 1 h at room temperature, and incubated overnight with primary antibodies (used at 1:100 dilution) at 4 °C. The next day, the samples were wash with PBST and incubated with secondary antibodies for 1 h at 37 °C: Alexa Fluor 546 donkey anti-rabbit IgG (used at 1:1000 dilution, #A10040, lifetech) or Alexa Fluor 488 donkey anti-mouse IgG (used at 1:1000 dilution, #A21202, lifetech). The nucleus was stained with DAPI (#F6057, Sigma-Aldrich, USA). Images were acquired by laser scanning confocal microscope. The Immunofluorescence colocalization analysis was performed with scatter J (a plugin for ImageJ), the presence of colocalization is defined as pearson’s coefficient value > 0.5.

MI-2 and mepazine (chemical structure shown in Figure 1A, synthetic compounds provided by Eternity Bioscience Inc. NJ, USA) was dissolved at a concentration of 30 mM in 100% DMSO as a stock solution, stored at −20°C, and diluted with medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study (all the control groups are composed of 0.1% DMSO). Cyclosporine A (CsA), phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and adenosine triphosphate (ATP) were purchased from Sigma-Aldrich (St. Louis, MO). Dextran sulfate sodium (DSS, 36–50 kDa) was bought from MP Biomedicals (Aurora, OH). Myeloperoxidase (MPO) activity assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RPMI-1640, FBS, Alexa Fluor 546 Donkey Anti-Rabbit IgG and Alexa Fluor^® 488 Donkey Anti-Mouse IgG (H+L) were purchased from Life technology (Carlsbad, CA). Anti-phospho-IκBα, anti-phospho-IKKα/β, were purchased from Cell Signaling Technology (Beverly, MA). Anti-NLRP3, anti-phospho-p65 and anti-CASP1 were purchased from Epitomics (Burlingame, CA). Anti-ASC and anti-COX2 were purchased from Santa Cruz (Santa Cruz, CA). ELISA kits for murine TNF, IL-1β, IL-6, IFN-γ and human IL-1β were purchased from Dakewe Biotech Co. Ltd (Beijing, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Brains were perfused and post-fixed with 4% paraformaldehyde at 4 °C for 12–16 h, cryoprotected in 30% sucrose and embedded in optimum cutting temperature compound (OCT). Coronal sections (20 μm thick) were obtained using a Leica cryostat (CM 3050S). Immunostaining was performed as previously described [11 (link)]. Chicken anti-GFP (Abcam, ab13970, 1:500) and rabbit anti-FOXG1 (Abcam, AB18259, 1:500) were used as primary antibodies, and Alexa Fluor 488 goat anti-chicken IgG (Molecular Probes, A11039, 1:500) and Alexa Fluor 546 donkey anti-rabbit IgG(Molecular Probes, A10040, 1:500) were used as secondary antibodies. DAPI (Sigma, D9564, 1:1000) was incubated for 15 min (mins) before coverslips were applied. Images were captured by a confocal microscope (Olympus, FV1000). For cell counting, three brains from each genotype from at least two different litters were collected and images of two consecutive hippocampal slices of each brain were used; the dorsal hippocampus was outlined using ImageJ software (NIH), and CA1 cell numbers were counted manually based on the DAPI staining.