Rat anti tubulin antibody

To investigate the regulation of rTsgal and GM on macrophage polarization, the RAW264.7 and murine peritoneal macrophages were used for the in vitro stimulation of M1 and M2 phenotypes. The cells (2 × 10⁶ cells/well) were stimulated using 20 μg/mL rTsgal or 1.6% GM at 37 °C for 24 h, soluble cell proteins were extracted using ice-cold cell lysis buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell protein concentration was measured using a BCA assay kit (Solarbio, Beijing, China) [24 (link), 69 (link)]. The expression level of M1 (iNOS)/M2 (Arg1) marker proteins in macrophages was then detected by Western blotting. Rabbit anti-iNOS antibody (1:1000; Abcam, Shanghai, China), mouse anti-Arg1 antibody (1:1000; Proteintech, Rosemont, IL, USA) and rat anti-tubulin antibody (1:5000; Abcam) were used as the primary antibodies; HRP-labeled goat anti-rabbit IgG, goat anti-mouse and goat anti-rat IgG (1:10000; Southern Biotech) were used as the second antibodies [32 (link), 59 (link)].

HDFs were cultured in 15-mm diameter confocal petri dishes (NEST, China) overnight. After treatments, cells were fixed with 4% paraformaldehyde for 20 min. Then the cells were washed twice with PBS before blocking in immunostaining blocking buffer (Cat# P0102, Beyotime, China) for 1 h. After incubation with rat anti-tubulin antibody (Cat# ab6160, Abcam, United Kingdom) diluted in 1% bovine serum albumin (BSA) in PBS overnight at 4°C, the dishes were washed with PBS for three times and then stained with Alexa Fluor 488 fluorescent secondary antibody (Cat# A32731, Invitrogen, United States) at 37°C for 1 h in the dark. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Cat# C1005, Beyotime, China), and the plates were sealed with anti-fluorescence quenching solution (Cat# P0126, Beyotime, China) before imaging. The pictures were acquired using a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).

After 8 or 12 h of culture, MI- or MII-stage oocytes were isolated for the immunofluorescence assay, to assess chromosome alignment or to detect the anti-CENP-A antibody signal in oocyte from serum-ACA-positive mice.
The procedures for the immunofluorescence assay were as follows: oocytes were fixed in 4% polyoxymethylene and permeated with 0.5% Triton X-100 (Sigma, USA), followed by sealing in 5% normal donkey serum (Jackson Immunoresearch, USA). For staining of Spindle microtubules, oocytes were incubated overnight at 4 °C with rat anti-tubulin antibody (Abcam, United Kingdom, 1:800 dilution). After three washes in washing buffer, oocytes were incubated with Alexa Fluor® 488 AffiniPure Donkey anti-Rat IgG (Jackson Immunoresearch, USA, 1:500 dilution) for 1 h at room temperature, rinsed, incubated with 1 μg/mL DAPI (Cell Signaling Technology, USA) for 15 min, rinsed again, and fixed in a dish for subsequent microscopic observation. For intra-oocyte IgG staining, following incubation with Alexa Fluor® 647 AffiniPure Donkey anti-Mouse IgG (Jackson Immunoresearch, USA, 1:500 dilution) for 2 h at room temperature, oocytes were washed 3 times in washing buffer, and co-stained with 1 μg/mL DAPI for 15 min. These oocytes were mounted on glass slides and examined with a confocal laser-scanning microscope (LSM780; Zeiss GmbH, Germany).