Mouse monoclonal anti α sma

The primary antibodies used were as follows: mouse monoclonal anti-α-SMA (clone: 1A4; dilution: 1:200), rabbit monoclonal anti-tenascin-C (clone: EPR4219; dilution: 1:200), mouse monoclonal anti-cytokeratin (clone: M20; dilution: 1:200), rabbit monoclonal anti-vimentin (clone: EPR3776; dilution: 1:200), and rabbit monoclonal anti-desmin (clone: Y66; dilution: 1:200), all from Abcam (Cambridge, UK). For immunochemical staining, biotinylated horse anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG (dilution: 1:100; Zhongshan Biotechnology, Beijing, China) were used as secondary antibodies. The reaction products were visualized in brown using a diaminobenzidine substrate kit (DAB; Zhongshan Biotechnology, Beijing, China). Counterstaining was performed with haematoxylin or methyl green. For immunofluorescence, Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 594 donkey anti-mouse IgG (dilution: 1:500; Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. diamidino-phenyl-indole (DAPI; dilution: 1:500; Invitrogen, Carlsbad, CA, USA) was used to stain the nuclei. Control staining was performed by substituting non-immune serum (or phosphate-buffered saline) for the primary antibodies. Fluorescence images were acquired using an inverted fluorescence microscope (IX71; Olympus, Tokyo, Japan).

The cells grown on cover slips were fixed with 4% paraformaldehyde for 10 min at room temperature. Following permeabilization with 0.5% Triton-X100 for 10 min, the fixed cells were blocked with 0.5% bovine serum albumin (Solarbio, Beijing, China) for 30 min and then incubated overnight at 4°C with the following primary antibodies: Mouse monoclonal anti-vimentin (1:500; cat. no. ab8978; Abcam), mouse monoclonal anti-α-SMA(1:500; cat. no. BM0001 Boster Biological Technology, Wuhan, China) and rabbit polyclonal anti-type I collagen (1:200; cat. no. BM0325 Boster Biological Technology). The immunoreactions were revealed using specific anti-mouse fluorescein isothiocyanate-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (1:200; cat. no. SA00003-1; Boster Biological Technology) and anti-rabbit Alexa Fluor 594-conjugated Goat Anti-Rabbit IgG Alexa Fluor-conjugated IgG (H+L) (1:200; cat. no. SA00006-4; Proteintech, Wuhan, China) for 1 h at room temperature. Negative controls were included by omitting the primary antibodies. Following washing with PBS, the immunolabeled cells were observed under a fluorescence microscope (Leica Microsystems, Mannheim, Germany). Densitometric analyses of the intensities of the vimintin, α-SMA and collagen I fluorescent signals were performed on digitized images using Image J software.

Double immunofluorescence labeling for podocalyxin, ED-1, E-cadherin and α-smooth muscle actin (α-SMA) was performed on paraffin-embedded kidney sections to label the podocytes, macrophages, tubular epithelial cells and myofibroblasts in the kidney. Sections (4 µm) pre-treated using heat-induced antigen retrieval were stained with polyclonal rabbit anti-podocalyxin (1:50; cat. no. ab205350; Abcam, Cambridge, MA, USA), monoclonal mouse anti-ED-1 (1:50; cat. no. MAB1435; Chemicom, Toronto, ON, Canada), polyclonal rabbit anti-E-cadherin (1:50; cat. no. ab53226; Abcam) and monoclonal mouse anti-α-SMA (1:50; cat. no. ab7817; Abcam) overnight at 4°C. They were then incubated with fluorescein isothiocyanate and rhodamine-conjugated goat anti-mouse (cat. no. sc-3796) and goat anti-rabbit (cat. no. sc-3839) secondary antibodies at 1:100 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Negative control was performed by replacing the primary antibody with phosphate-buffered saline. To stain the nuclei, 4′,6-diamidino-2-phenylindole (Bioss, Inc., Beijing, China) was used. The sections were examined by a fluorescence microscope (Nikon CE1 Confocal Microscope, Nikon, Tokyo, Japan). Renal tissues with positive double immunofluorescence staining were used for future 2-DE. Negative tissues were abandoned.