Rabbit monoclonal anti vimentin

RPE cells were seeded on glass coverslips (12 mm diameter) to confluence in a 24-well plate. Cells were serum starved for 2 days before adding TGFβ2 for 72 h. Cells were rinsed in PBS ×2 before fixation in 4% paraformaldehyde for 10 min and then rinsed in PBS (3 × 5 min). Cells were permeabilized using 0.01% Triton X-100 in PBS for 5 min and then rinsed in PBS (3 × 5 min). Cells were blocked for 1 h at RT in 1% bovine serum albumin and 3% normal goat serum diluted in PBS. Cells were incubated in the primary antibody overnight at 4 °C (rabbit monoclonal anti-vimentin at 1:200 from Cell Signaling Technology #5741S and mouse monoclonal anti-alpha smooth muscle actin at 1:400 from Sigma #A2547). The next day, cells were washed in PBS (3 × 10 min) and incubated for 2 h in the dark at RT in the secondary antibody: goat anti-rabbit Alexa Fluor 488 (1:1000; Invitrogen #A11034) and/or goat anti-mouse Alex Fluor 594 (1:1000; Invitrogen #A11032) and DAPI (1:100). Cells were rinsed in PBS (3 × 10 min). Coverslips were mounted on slides using mounting medium (ibidi #50011) and sealed with CoverGrip Coverslip Sealant (Biotium #23005). Z-stack images were taken on the Leica SP8 confocal microscope and processed using FIJI and Adobe Photoshop.

For immunofluorescence, cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 30 min. Lung tissues fixed in 4% paraformaldehyde for 48 h were embedded in paraffin and cut into 4-μm thick sections. Non-specific binding was blocked using 10% normal goat serum (Sigma). Cells or lung sections were incubated with the following primary antibodies after being diluted in PBS with 1% bovine serum albumin at 4°C overnight: mouse monoclonal anti E-cadherin (Cell Signaling Technology, Inc.) and rabbit monoclonal anti-Vimentin (Cell Signaling Technology, Inc.). Then, cells or tumor sections were washed twice with PBS and incubated with secondary antibodies at 37°C for 30 min as follows: FITC-conjugated goat-anti-rabbit IgG (Abcam) or TRITC-conjugated goat-anti-mouse IgG (Sigma). The slides were mounted in mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and viewed with a live cell station (Delta Vision, API).

Western blot analyses were conducted according to the method described previously [12 (link)]. The primary antibodies were: rabbit monoclonal anti-E-cadherin (#3195, 1:1000, Cell Signaling Technology), rabbit monoclonal anti-Vimentin (#5741, 1:1000, Cell Signaling Technology), rabbit polyclonal anti-UPF1 (23379-1-AP, 1:1000, ProteinTech), rabbit polyclonal anti-FOXQ1 (23718-1-AP, 1:1000, ProteinTech), mouse monoclonal anti-β-actin (A5316, 1:5000, Sigma-Aldrich), and mouse monoclonal anti-PCNA (sc-25280, 1:1000, Santa Cruz). The blot signal was detected using ECL (GE Healthcare, UK).

Western blot analysis was conducted as previously described [23 (link)]. The primary antibodies were used as follows: rabbit polyclonal anti-LATS2 (1 : 500, Proteintech, Rosemont, USA), rabbit polyclonal anti-YAP (1 : 500, Proteintech), rabbit monoclonal anti-phospho-YAP (Ser127, 1 : 1000, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-E-cadherin (1 : 1000, Cell Signaling Technology), rabbit monoclonal anti-Vimentin (1 : 1000, Cell Signaling Technology), and rabbit polyclonal anti-MMP9 (1 : 500, Proteintech). Mouse monoclonal anti-β-actin (1 : 1000, Sigma-Aldrich) was used as a reference.