Goat anti rabbit igg h l

Twenty oocytes in each group were fixed with Immunol Staining Fix Solution (P0098, Beyotime, Shanghai, China) for 15 min at room temperature. They were then permeabilized with 1% TritonX-100 (P0096, Beyotime, Shanghai, China) for 20 min and incubated with immunofluorescence-blocking solution (P0228, Beyotime, Shanghai, China) for 30 min. The oocytes were subsequently incubated with Caspase-3 (A0214, Abclonal, Wuhan, China) antibody, diluted with QuickBlock™ Immunostaining Primary Antibody Dilution Solution (P0262, Beyotime, Shanghai, China), at 1:100, for 12 h at 4 °C. The oocytes were incubated with goat anti-rabbit IgG(H + L) (A0214, Beyotime, Shanghai, China), diluted with Immunol Fluorescence Staining Secondary Antibody Dilution Buffer (P0108, Beyotime, Shanghai, China), at a 1:200 ratio for 1 h at room temperature. After incubation, the oocytes were stained with Hochest 33342 for 10 min, and then washed with DPBS 3 times. The samples were observed under a Nikon eclipse Ti-S microscope (ti-2U, Nikon, Tokyo, Japan).

Cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After that, samples were washed with PBS three times, for 3 min each time, at 25°C and blocked with 1% BSA (Solarbio, Beijing, China) for 1 h at room temperature. Subsequently, cells were incubated with rabbit anti-γ-H2AX antibody (ab2893, Abcam, UK) in PBS overnight at 4°C followed by goat anti-rabbit IgG (H+L) (A0423, Beyotime, Haimen, China) for 1 h at room temperature. Images were obtained by an ECLIPSE Ni microscope and a digital image analyzer (Nikon, Tokyo, Japan).

A polyclonal anti-c-Maf antibody was obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. An anti-MafB antibody was purchased from Abgent (Suzhou, China). Monoclonal anti-HA, anti-Myc, anti-Flag, and anti-GAPDH antibodies were obtained from MBL (Tokyo, Japan). An anti-USP5 antibody was purchased from Proteintech (Chicago, IL, USA). MG132 was purchased from Santa Cruz Biotechnology, and cycloheximide (CHX) was purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). WP1130 was purchased from Selleck Chemicals Inc. (Houston, TX, USA); HRP-labeled goat anti-mouse and goat anti-rabbit IgG (H+L) antibodies were purchased from Beyotime Institute of Biotechnology (Nantong, China). IRDye 680CW goat anti-mouse and IRDye 800CW goat anti-rabbit antibodies were from Odyssey (San Ramon, CA, USA).

A375 and MV3 melanoma cell lines were harvested, then suspended in RIPA Lysis Buffer. Protein concentrations were detected with BCA protein assay kit (Beyotime Biotech, China). The lysates from cells as well as the fresh tissues were separated by SDS-PAGE, followed by transferring onto PVDF membranes (Millipore, USA). Followed by blocking with 5% BSA, the PVDF membranes were incubated gently with a primary antibody against human GAPDH (1:1000, Beyotime), p21 (1:1000, Cell Signaling), CDK2 (1:1000, Cell Signaling), Cyclin E (1:1000, Cell Signaling), E-cadherin (1:1000, Cell Signaling) and vimentin (1:1000, Cell Signaling) at 4°C overnight, followed by appropriate (horseradish peroxidase-conjugated secondary antibody) HRP-conjugated secondary antibodies. HRP-labeled goat anti-mouse IgG (H + L) (A0216, 1:2000) and goat anti-rabbit IgG (H + L) (A0208 1:2000) were used as secondary antibodies which purchased from Beyotime. Proteins were visualized by ECL Western blot analysis system. The signal was captured by the ECL reagent (Beyotime) and visualized by Western blotting detection instruments (Clinx Science).

Proteins were purified in RIPA lysis buffer containing a protease and phosphatase inhibitor mixture (Roche Diagnostics). Protein concentrations were tested via a BCA assay kit (Pierce Diagnostics). Proteins were separated on SDS/PAGE gels, transferred to polyvinylidene difluoride membranes (Millipore), blocked in 5% skim milk (OXOID) in TBST (0.02 M Tris base, 0.1% Tween 20, 0.14 M NaCl pH 7.4), and incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti-uncoupling protein 1 (UCP1) (ab209483, Abcam) and anti-HSP90 (4874, Cell Signaling Technology). Goat anti-Rabbit IgG H&L (A0277, Beyotime). The primary antibody was diluted at a ratio 1:1000; The secondary antibody was diluted at a ratio 1:5000. Signals were detected with super signal west pico chemiluminescent substrate (Pierce). Intensity values of the bands were analyzed via ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Tumor tissue sections were fixed with 10% paraformaldehyde, embedded in paraffin, sectioned at a thickness of 4 μm, and stained using a standard hematoxylin and eosin technique. Paraffin sections were also immunostained with antibodies specific for MSLN (ZSGB-BIO, Beijing, China) overnight at 4 °C, followed by secondary staining with goat anti-rabbit Ig (PV-9000) (ZSGB-BIO, Beijing, China). Images of all sections were obtained with a microscope (DMI6000B; Leica Microsystems, Wetzlar, Germany).
For the immunofluorescence imaging, paraffin sections were incubated with antibodies specific for CD3 (ZSGB-BIO, Beijing, China) overnight at 4 °C, followed by secondary staining with goat anti-rabbit IgG (H + L) (Beyotime, Shanghai, China). Images of sections were obtained with a laser scanning confocal microscopy (LSM 800, Carl Zeiss AG, Oberkochen, Germany).