Cy3 conjugated α sma antibody

Paraffin-embedded tissue sections were deparaffinized, and antigen retrieval was performed using boiling 0.01M citrate buffer (pH 6) for 20 min. Sections were blocked with 5% goat serum in Dako serum-free blocking reagent for 30 min. For α-smooth muscle actin (α-SMA) staining, sections were incubated overnight at 4°C with Cy3-conjugated α-SMA antibody (1:400)(Sigma-Aldrich, Oakville, Ontario, Canada). After washing with PBS-0.1% Tween, sections were counterstained with DAPI and mounted with Slow Fade mounting medium (Invitrogen, Waltham, MA, USA). For CD3/B220 staining, the sections were incubated overnight at 4°C with CD3 antibody (1:200)(DAKO, Mississauga, Ontario, Canada) and Biotin conjugated B220 antibody (1:250)(BD, Mississauga, Ontario, Canada). After washing, sections were incubated with goat anti-rabbit IgG Alexa Fluor^® 555 and Alexa Fluor^® 488 streptavidin (Molecular Probes, Eugene, OR) at the dilution of 1:400. Antibodies for myeloperoxidase (MPO) (1:400) and F4/80 (1:500)(Abcam, Cambridge, MA, USA) were used for neutrophil and macrophage staining, respectively. Goat anti-rabbit IgG Alexa Fluor^® 555 (Molecular Probes, Eugene, OR, USA) were used as the secondary antibody at the dilution of 1:400. Then the sections were counterstained with DAPI and mounted.

Immunostaining was performed on cultured BMDMs and frozen sections of human and mouse tissues fixed by 2% paraformaldehyde following a previously described protocol.^[18 ,
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^] In brief, sections were labeled overnight with various combinations of directly conjugated primary antibodies as follows: fluorescein isothiocyanate (FITC)‐conjugated anti‐CD68 antibody (sc‐20060 FITC, Santa‐cruz), FITC‐conjugated anti‐F4/80 (11‐4801‐81, eBioscience), Cy3‐conjugated α‐SMA antibody (C6198, Sigma), Runx1 (sc‐365644, Santa Cruz), p‐Runx1(Ser397) (PA5‐105609, Invitrogen) detected with APC (A21240, Invitrogen) or PE‐conjugated secondary antibodies (A11003, Invitrogen). Sections were washed and, in some cases, DNA was counterstained with DAPI and observed under a fluorescence microscope (Carl Zeiss Axio Observer Z1) or confocal microscope (Carl Zeiss LSM 880).

The frozen kidney tissues were embedded with OCT and cut into 5 μm-thick sections and stored at -80°C until use. After the frozen sections were dried, they were fixed with acetone, rinsed three times with PBS, blocked with goat serum for 1 h, and then incubated with primary antibody (fibronectin antibody and collagen I antibody, Abcam) overnight at 4°C. After washing with PBS, the Alexa Fluor 488 was applied for 1 h. For α-SMA staining, kidney tissues were cut into 4 μm-thick sections after embedding in paraffin, which were subjected to antigen retrieval, washed with PBS, blocked, and incubated overnight with Cy3-conjugated α-SMA antibody (Sigma). Frozen sections were incubated overnight with primary CD45 (Bioscience) or CD11b (Bio-Rad) or CD206 (Bio-Rad) and α-SMA (Abcam) antibodies for double immunofluorescence staining, and then followed by applied with Alexa Fluor 488 or 647. Sections were mounted with DAPI and mounting medium. Images were obtained from fluorescence microscope (Nikon, Japan) and Zeiss confocal microscope (oberkochen, Germany).