Alexa 594 conjugated anti rabbit igg

Mouse PaSCs from adherent cultures were digested with 0.25% trypsin/EDTA and centrifuged at 800 rpm for 3 min. The cell pellets were resuspended in complete medium. After preparing 6-well plates with coverslips, cell suspension was added into each well. The cells were cultured at 37 °C in 5% CO₂ for 48 h, washed with PBS and fixed with 4% paraformaldehyde (PFA) (ZSGB-BIO, Beijing, China) for 15 min. Then, cells were treated with 10% donkey serum at room temperature for 1 h and incubated with the following primary antibodies: mouse monoclonal anti-α-SMA (1:100), rabbit polyclonal anti-fibronectin (1:50), and rabbit polyclonal anti-collagen I (1:50) at 4 °C overnight. The cells were then incubated with Alexa 594-conjugated anti-rabbit IgG (Invitrogen, Chicago, USA) (1:1000) or Alexa 488-conjugated anti-mouse IgG (Invitrogen, Chicago, USA) (1:1000) for 1 h at room temperature. Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Munich, Germany) for 5 min. The stained coverslips were visualized using a Nikon 80i fluorescence microscope.

Immunofluorescence was performed as previously described (16 (link)). Cells were cultured on coverslips overnight and fixed with 4% paraformaldehyde for 20 min at room temperature. Samples were then incubated with 0.3% Triton X-100 for 10 min and blocked for 2 h with the blocking solution (Beyotime), after which cells were probed overnight at 4°C with a diluted primary antibody, followed by a secondary antibody for 2 h. The primary antibodies used were: a mouse monoclonal antibody for Pan-ck (1:300, Abcam), Vimentin (1:150, Santa Cruz), and rabbit polyclonal antibody against E-cadherin (1:100, Abcam), α-SMA (1:100, Abcam), and Smad5 (1:100, Abcam); the secondary antibodies used were Alexa 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) and Alexa 594-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA).

Tissue sections were stained with antibodies to IgG (Bethyl lab) and nephrin (Abcam) for 16 hours at 4°C followed by staining with Alexa594 conjugated anti-rabbit IgG, Alexa488 conjugated anti-guinea pig IgG, and Alex594 conjugated anti-phalloidin (Invitrogen). Nuclei were stained with 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen). Isotype-control staining was conducted via probing with rabbit IgG rather than with primary antibodies. Confocal images were acquired using an LSM 700 confocal microscope (Zeiss).

C8-D1a cells (2 × 10⁴ cells per cm²) and Neuro-2A cells (2 × 10⁴ cells per cm²) were seeded on poly-d-lysine (PDL) coated cover slips in culture plates. Cells were fixed in 2% paraformaldehyde (Sigma) for 10 min and then incubated with primary AQP4, p-NF-κB p65, EAAT2, SYP and PSD-95 antibodies in gelatin detergent buffer (GDB) [0.1% gelatin (Sigma Aldrich), 0.3% Triton X-100 (Thermo Scientific), 16 mM sodium phosphate (Sigma Aldrich), 450 mM NaCl with pH 7.4 (Sigma Aldrich)] at 4 °C overnight. Cells were washed three times with 1 × PBS and incubated with secondary Alexa 488-conjugated anti-mouse IgG (Invitrogen) and Alexa 594-conjugated anti-rabbit IgG (Invitrogen) antibodies for one hour at room temperature. Cells were counterstained using mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI, Thermo Scientific). Cells were visualized using a K1-Fluo confocal microscope (Nanoscope systems) and fluorescence intensity were analyzed using ImageJ software.

The cells cultured for 9 days were fixed with 2% paraformaldehyde in PBS (pH 7.4) for 60 min at room temperature. The fixed cells were washed two times with PBS and permeabilized with TRITON X-100 (ICN Biomedical, Irvine, CA, USA) in PBS containing 0.1% BSA for 15 min and then blocked with 10% normal serum from the same animal species as the secondary antibody for 10 min. Next, the samples were incubated overnight at 4 °C with rabbit anti-AFP antibody (Abcam, Cambridge, UK) diluted 1:500, goat anti-α1-antitrypsin antibody (Abcam) diluted 1:300, rabbit anti-glial fibrillary acidic protein antibody (Sigma-Aldrich Co, St. Louis, MO, USA) diluted 1:80, or rabbit anti-CNPase antibody (Abcam) diluted 1:100 in PBS containing 1% BSA. After washing three times with PBS, the samples were incubated with FITC-conjugated anti-goat IgG (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and Alexa 594-conjugated anti-rabbit IgG (Invitrogen Co, Carlsbad, CA, USA) as secondary antibodies for 1 h. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Then, the cells were analyzed using fluorescence microscopy.

Cells were labeled with 4 μg/ml BrdU for approximately 18 hours. After washing with PBS cells were fixed with 75% ethanol (−20°C) for 15 min. Subsequently, cells were treated with 1 M HCl for 1 h at 37°C. They were then treated twice with sodium borate for 5 min. After incubation with 10% BSA in PBS for 20 minutes at room temperature they were incubated with a mouse anti-BrdU antibody (1:20; BD Biosciences) at room temperature for 1 h. PBS washes were performed between each step. Cells were incubated with anti-mouse conjugated to Alexa 488 (1:400, Invitrogen) for 1 h. The nuclei were counterstained with DAPI. For combination with p21 staining, cells were incubated after sodium borate treatment with mouse anti-BrdU antibody (as above) plus rabbit anti-p21 antibody (1:100; Cell Signaling, #2947) at room temperature for 1 h, followed by Alexa 488-conjugated anti-mouse IgG (1:400, Invitrogen) and Alexa 594-conjugated anti-rabbit IgG (1:400, Invitrogen) for 1 h.