Rabbit polyclonal anti tgf β1 antibody

Histology and immunohistochemical staining of 4-μm-thick tissue sections were performed as described previously [22 (link)]. The following primary antibodies were used: rabbit polyclonal anti-G9a antibody (Abcam, Cambridge, UK), mouse monoclonal anti-α-smooth muscle actin (SMA) antibody (Sigma-Aldrich), rabbit polyclonal anti-collagen I antibody (Abcam), rabbit polyclonal anti-collagen III antibody (Abcam), rat monoclonal anti-mouse CD68 antibody (Serotec, Oxford, UK), rabbit polyclonal anti-TGF-β1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-H3K9me1 antibody (Abcam).
The numbers of cells positive for α-SMA, CD68, TGF-β1, and H3K9me1 in the submesothelial compact zone were counted in 10 fields at ×200 magnification. Areas containing collagen I and III were assessed at ×200 magnification in predetermined fields of the submesothelial compact zone captured by a digital camera and analyzed using ImageJ software (version 1.48p; National Institutes of Health, Bethesda, MD, USA) in 10 fields.

Kidney samples (20 mg) were homogenized in RIPA lysis buffer containing complete protease inhibitor cocktail (Roche). Protein concentration was determined by the BCA Assay. Samples were mixed with 2x Laemmli buffer and boiled. Equal amounts of protein (40 µg) were separated on 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes and blocked with 5% skim milk in Tris-buffered saline (TBS), containing 0.1% Tween-20. Membranes were incubated overnight at 4 °C with primary antibodies (rabbit polyclonal anti-TGF-β₁ antibody 1:500, SantaCruz Biotechnology, Santa Cruz, CA, USA; rabbit polyclonal anti-PKG 1:2000, Enzo, Farmingdale, NY, USA; rabbit polyclonal anti-sGC_ß1 1:2000, Novus Biologicals; rabbit polyclonal anti-PDE5a 1:1000, Enzo; p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (pERK1/2) (Thr202/Tyr204) both at 1:1000, Cell Signaling, Danvers, MA, USA), then washed and incubated with peroxidase-conjugated secondary antibody (anti-mouse IgG or anti-rabbit IgG, 1:2000, Cell Signaling). Blots were visualized by ECL detection kit (Pierce Thermo). Original immunoblots are shown in Supplementary Figure 2 for PDE-5, TGF-ß1 and p-ERK and in Supplementary Figure 3 for PKG and sGC_ß1.

Histologic and immunohistochemical staining of 4-μm-thick tissue sections was performed as previously described [28 (link), 29 (link)]. The following primary antibodies were used: mouse monoclonal anti-α-SMA antibody (Sigma-Aldrich), rabbit polyclonal anti-FSP-1 antibody (Abcam, Cambridge, UK), rabbit polyclonal anti-collagen I antibody (Abcam), rabbit polyclonal anti-collagen III antibody (Abcam), rabbit polyclonal anti-TGF-β1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-SET7/9 antibody (Abcam), and rabbit polyclonal anti-H3K4me1 antibody (Abcam).
Areas that contained collagens I or III were assessed in predetermined fields (×200 magnification) of the submesothelial compact zone, captured by a digital camera and analyzed using ImageJ software (version 1.48p; National Institutes of Health, Bethesda, MD, USA) in 10 fields. We counted cells expressing α-SMA, FSP-1, TGF-β1, SET7/9 or H3K4me1 in the submesothelial compact zone in 10 fields at ×200 magnification.