Alexa fluor 488 conjugated anti rabbit igg

Arterial tissue samples were first washed with cold PBS and then fixed with 4% paraformaldehyde at 4°C. The samples were processed by successive incubation in (1) PBS for 20 min; (2) an ethanol series (70%, 85%, 95%, 95%, 100%, and 100%) for 1 h at room temperature; (3) butyl alcohol, three times for 30 min each at room temperature; and (4) fresh paraffin at 65°C, three times for 30 min each. The treated samples were embedded in paraffin, and 5-μm-thick sections were prepared. After the sections were dewaxed and rehydrated, immunofluorescence was performed according to standard protocols. Briefly, sections were incubated with mouse anti-α-SMA (cat No. ab32575, 1:200; Abcam) or rabbit anti-PH3 (cat No. 53348; Cell Signaling Technology) overnight at 4°C. After being washed with PBS three times, sections were incubated with Alexa Fluor 647-conjugated anti-mouse IgG (cat No. 4410S; Cell Signaling Technology) or Alexa Fluor 488-conjugated anti-rabbit IgG (cat No. 4416S; Cell Signaling Technology) for 30 min at room temperature. Slices were then stained with DAPI (1.0 mg/ml; Invitrogen) for 30 min before mounting. Images were captured with an Olympus confocal microscope (Olympus, Japan).

Cells were seeded on Millicell EZ 4-well glass slides (Millipore, MA, USA). After treatment for indicated time, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA). Cells were then incubated with primary antibodies at 4°C overnight, and secondary antibody for 1 hour at room temperature. The sources and dilutions of antibodies are: mouse anti-α-SMA (1:50, Abcam), rabbit anti-fibronectin (1:200, Abcam), rabbit anti-Vimentin (1:100, Cell Signaling Technology), Alexa Fluor 555-conjugated anti-mouse IgG (1:1000, Cell Signaling Technology), Alexa Fluor 555-conjugated anti-rabbit IgG (1:1000, Cell Signaling Technology), and Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000, Cell Signaling Technology). Cell nuclei were stained with 50 ng/ml 4’,6-diamidino-2-phenylindole (DAPI) for 5 min. Slides were mounted with anti-fade fluorescent mounting medium (Applygen, #C1210). Images were acquired by a Zeiss LSM 510 confocal laser scanning microscope (CLSM, Carl Zeiss, Germany) and processed by Adobe Photoshop CS6.