Sc 11769

Localization of endogenous pSMAD2/3 and PDCD4 was assessed by immunofluorescent staining. U251 GBM cells were seeded on 4 well chamber slides (Lab-Tek.,Thermo Fischer Scientific) . After attachment and treatment as indicated, cells were fixed in 4% paraformaldehyde (in PBS +0.1% Triton X-100 for permeabilisation) (pH 7.4,) and then blocked with 3% BSA in PBS, followed by overnight incubation with goat polyclonal-anti- pSMAD2/3 (sc-11769, (Santa Cruz Biotechnology, Santa Cruz, CA)) and rabbit-anti-PDCD4 primary antibody (#9535, CST) (1:500 dilution with PBS containing 3% BSA). Alexa flour conjugated AffiniPure donkey anti-goat IgG and goat-anti-rabbit IgG secondary antibodies (Invitrogen) were then applied. Slides were then mounted with fluorescent medium VECTASHIELD containing DAPI (Nuclear stain) (Vector Laboratories Inc., Burlingame, CA) to preserve the fluorescent signal. The localization of endogenous pSMAD2/3 and PDCD4 were visualized and images captured with Olympus FSX100 fluorescent microscope.

Three µm sections were processed for immunohistochemistry as follows. Endogenous peroxidase was blocked with 3% hydrogen peroxide and immunohistochemical staining for fibronectin (F3648, 1∶100 dilution; Sigma-Aldrich, St Louis, MO, USA), collagen I (Ab21286, 1∶800; Abcam, MA, USA), α-SMA (A2547, 1∶1,000; Sigma-Aldrich, St Louis, MO, USA and NCL-MSA 1∶200; Leica Microsistemas, Barcelona, Spain), phospho-Smad2/3 (sc-11769, 1∶50 dilution; Santa Cruz Biotechnology Inc, CA, USA) and phospho-Smad1 (Ab63439, 1∶50 dilution; Abcam, Cambridge, UK) were performed. Then, sections were washed three times in PBS and incubated with the Novolink Polymer Detection System (Novocastra, MA, USA), followed by reaction with 3,3′diaminobenzidine as chromogen. Negative WT slides were prepared without primary antibody.

The apex of the heart tissue was fixed with 4% phosphate buffered saline (PBS) buffered paraformaldehyde, embedded in paraffin, and then sectioned into 3 μm thicknesses and stained with Masson trichrome according to the standard procedure. Masson trichrome staining causes muscle fibers to turn red and collagen fibers to turn blue. Immunohistochemistry (IHC) staining was used to detect the distribution and expression of biomarkers for fibrosis. We used a microwave-based heating antigen retrieval and 3% H₂O₂ to block non-specific staining. The antibodies used included: collagen I (1310-01, SouthernBiotech, Birmingham, AL, USA; 1:200 dilution), collagen III (GB111629, Servicebio, Wuhan, China; 1:200 dilution), α-SMA (GM085102, Gene Tech, Shanghai, China; 1:200 dilution), phosphorylated Smad2/3 (sc-11769, Santa Cruz Biotechnology, Dallas, TX, USA; 1:200 dilution), and anti-β-catenin (610154, BD Transduction Laboratories, Shanghai, China; 1:50 dilution). Images were quantitatively analyzed using Image-Pro Plus v 6.0 (Media Cybernetics, Rockville, MD, USA). Briefly, the accumulation of collagens I and III and α-SMA in 10 random areas, except staining around blood vessels, was analyzed under 200× for the percentage of positive areas in the examined field. Numbers of p-Smad2/3+ cells were counted in 10 random areas for each sample under 400×.

Knee joints were dissected from mice, the attached muscle was carefully removed and the tissues were fixed overnight with 10% formalin at 4 °C. After washing three times with ice-cold PBS, the samples were decalcified at 4 °C using 10% EDTA (pH 7.4) for 21 days and then embedded in paraffin. Four-micrometer-thick sagittal sections of the medial compartment of the knee were used for staining. The sections were stained with individual primary antibodies against COX-2 (ab15191, 1:100, Abcam), osterix (ab22552, 1:100, Abcam), OCN (M137, 1:100, Takara Bio Inc., Kusatsu, Shiga Prefecture, Japan), pSmad2/3 (sc-11769, 1:50, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and MMP13 (ab3208, 1:50, Abcam) at 4 °C overnight. We used the horseradish peroxidase–streptavidin detection system (Dako) to detect the immunoreactivity. Then, we counterstained the sections with hematoxylin (Sigma-Aldrich). We counted the number of positively stained cells in four random visual fields of the subchondral bone in five sequential sections from each mouse in each group. For OCN staining, we normalized the number of positively stained cells to the number of cells per millimeter of adjacent bone surface (N·mm⁻¹) in subchondral bone.

Immunohistochemical analyses of sections of each construct were performed using an Anti-Rabbit/Mouse HRP-DAB Cell & Tissue Staining Kit (R&D Systems, USA). Sections were subjected to epitope recovery in citrate buffer at 99 °C for 30 min. Once room temperature was reached, slides were washed in triethanolamine-buffered saline, and nonspecific immunoglobulin binding was blocked with 5% (V/V) bovine serum albumin for 30 min at room temperature. Sections were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Osterix (Santa Cruz Biotechnology, 1:100, USA); rabbit anti-Col X (Abcam, 1:100, ab58623) and a TGF-β pathway-specific antibody against p-Smad2/3 (sc-11769, Santa Cruz Biotechnology, 1:100, USA). All sections were incubated with a biotinylated secondary antibody, stained using an R&D HRP-DAB Staining Kit and counterstained with haematoxylin. After mounting, the slides were photographed with an Olympus BX53 microscope (Olympus, Japan). The numbers of Col X-positive cells in the cartilage layer, TRAP-positive osteoclasts, Osterix-positive cells and p-Smad2/3-positive cells in the posterior and middle condylar subchondral bone were determined. The percentages of positive cells in all chondrocytes are shown. All sections were placed onto one slide and processed together under the same conditions.