Anti alpha smooth muscle actin

Sodium hyaluronate (100kDa) was obtained from Lifecore Biomedical and stored at −20°C until use. Dimethylformamide (DMF), glycidyl methacrylate (GM), triethylamine (TEA), 2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (#410896), differentiation solution (A3179-1L), 2-methylbutane, picric acid solution (P6744), Fast Green FCF (F7252), and Direct Red 80 (#365548) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Spectrum^™ Spectra/Por^™ 3 RC dialysis membrane tubing, 3500 Dalton MWCO (#08-670-5B), Wheat Germ Agglutinin (WGA) Alexa Fluor 488 (#W11261), Hoescht (#33342), high concentration rat tail collagen type I (#CB354249), and NanoOrange Protein Quantitation Kit (#N6666) were purchased from Thermo Fisher Scientific (Waltham, MA). Protein LoBind microcentrifuge tubes (#022431102) were purchased from Eppendorf (Hamburg, Germany). OCT was obtained from Sakura Finetech USA, Inc. (Torrance, CA). Harris Hematoxylin Stain (#95057-858) and Eosin Y Solution (#95057-848) were purchased from VWR (Radnor, PA). Recombinant human TGF-β1 (#100 - 21) was obtained from PeproTech. Recombinant human decorin protein (ab167743), human decorin ELISA kit (ab99998), anti-decorin antibody (ab151988), anti-sarcomeric alpha actinin (ab137346), and anti-alpha smooth muscle actin (ab5694) were purchased from Abcam (Cambridge, UK).

Griess reagent, 5,5’-Dithiobis (2-nitrobenzoic acid) and Lucigenin were purchased from Sigma-Aldrich. Hydrogen peroxide 33% was procured from PanReac AppliChem. The following antibodies were used in this work: Anti-fibronectin was obtained from GeneTex. Anti-MMP-9 and anti-Bcl2 were obtained from Proteintech. Anti-vimentin, anti-MMP-3, Anti-Akt, Anti-p38, Anti-Cleaved PARP1, Anti-p53 (acetyl K382), Anti-JNK, Anti-alpha smooth muscle Actin, Anti-NF-kB p65 (phospho S536), and anti-β catenin were all obtained from Abcam (Cambridge, UK). Anti-cleaved caspase-3, anti-cleaved aspase-9, and anti-cleaved caspase-8 were obtained from Cell Signaling. Rabbit Polyclonal JNK1/2/3 was obtained from ORIGENE. Anti-beta Actin was obtained from Thermo Fisher Scientific. Peroxidase AffiniPure Goat Anti-Rabbit IgG and Peroxidase AffiniPure Goat Anti-Mouse IgG were obtained from Jackson ImmunoResearch Inc. The following ELISA kits were used in this work: nitrotyrosine ELISA kit (Abcam, ab113848), Hydrogen Peroxide Colorimetric/Fluorometric assay kit (Biovision, K265-200), malondialdehyde assay kits (Northwest Life Sciences Specialties, NWK-MDA01) and Superoxide Dismutase assay kit (Cayman chemical, 706002).

Patient tumor material or xenografts were fixed in 4% formalin prior to paraffin embedding. Sections of 5 μm were prepared on a microtome. Tissue sections were deparaffinized and heat mediated antigen retrieval was performed using Tris-EDTA buffer solution pH 9 for Hedgehog staining or 10 mM sodium citrate solution pH 6 for other stainings. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS. Aspecific staining was blocked using Ultra-V Block (Immunologic) for 10 min at room temperature. Primary antibodies were diluted in normal antibody diluent (KliniPath), applied on tissue sections and incubated overnight at 4°C in a humidified chamber. For amplification of signal Brightvision + post antibody block (Immunologic) was used prior to the addition of the secondary antibody; poly-HRP-anti Ms/Rt/Rb IgG (Immunologic) both for 30 min at room temperature. Visualization was performed using Vector® NovaRED™ (Vector Labs) according to manufacturer’s protocol, counterstained with 30% haematoxylin and tissue sections were mounted with non-aqueous medium. Antibodies used for immunohistochemistry were: anti-alpha smooth muscle actin (Abcam, 1:1000); anti-Hedgehog (clone H160, Santa Cruz, 1:1500), anti-Cytokeratin 19 (clone RCK108, BioGenex, 1:1000), anti-pan cytokeratin (clone AE1/AE3, Dako, 1:100).

Rats were euthanized using pentobarbital (100 mg/kg, intraperitoneal) and perfused intravascularly with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). Brains were harvested and sectioned into 18-μm-thick slices with a cryostat after embedding. Immunohistochemical and immunofluorescence studies were performed as previously described [13 (link)]. The primary antibodies were rabbit anti-HO-1 (1:400 dilution; Abcam, Cambridge, USA), goat anti-Iba-1 (1:400 dilution; Abcam), mouse anti-CD68 (1:100 dilution; Abcam), mouse anti-rat CD163(1:100 dilution; AbD Serotec, Hercules, USA), polyclonal rabbit anti-alpha smooth muscle actin (1:200 dilution; Abcam). The secondary antibody in the immunofluorescence studies was Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen, Carlsbad, USA). Nuclear labeling was performed using fluoroshield™ with DAPI (F6057). Negative controls were performed without primary antibodies.

Extracellular matrix expression of the renal cortex in cryostat sections was identified by fluorescent staining using a specific anti-fibronectin (Abcam), anti-collagen type IV (Abcam), anti-alpha-smooth muscle actin (Abcam), anti-Gr1 (BD Biosciences) antibody, following permeabilization with 0.05% saponin buffer using a Vectastain Elite ABC kit (Vector Lab, Inc., Burlingame, CA, USA) as immunoperoxidase. The slides were developed using AEC Chromogen/Substrate and counterstained with hematoxylin. Isotype and species-matched irrelevant antibodies served as controls.

Tissues or cells were fixed with 4% paraformaldehyde, stabilized in 0.2% Triton X-100 for 10 min until cell membrane rupture, washed with PBS 3 times, and immersed in 2% BSA for 30 min to inhibit nonspecific antigen binding sites. The tissues were then incubated with anti-trypsinogen (1:500, Abcam, Cambridge, UK, ab166898) antibodies and anti-alpha smooth muscle actin (1:500, Abcam, Cambridge, UK, ab124964) overnight at 4 °C. The cells were then incubated with anti-trypsinogen (1:500, Abcam, Cambridge, UK, ab166898) antibodies or anti-alpha smooth muscle actin (1:500, Abcam, Cambridge, UK, ab124964) overnight at 4 °C. After washing, the tissues or cells were incubated with secondary antibody (Invitrogen) for 60 min, and the nuclei were stained with DAPI (Invitrogen) for 2 min. Then, the cells were washed with PBS and shielded from light before observation with a fluorescence microscope.