The tumors were resected immediately after the last intravital microscopy examinations and fixed in phosphate-buffered 4% paraformaldehyde. Pimonidazole [1-[(2-hydroxy-3-piperidinyl)-propyl]-2-nitroimidazole] was administered as described previously and used as hypoxia marker [23] (link), and α-smooth muscle actin (α-SMA) was used as marker for pericytes. Immunohistochemistry was done by using a peroxidase-based indirect staining method [23] (link). An antipimonidazole rabbit polyclonal antibody (gift from Prof. J.A. Raleigh, Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC) or an anti–α-SMA rabbit polyclonal antibody (Abcam, Cambridge, United Kingdom) was used as primary antibody, diaminobenzidine was used as chromogen, and hematoxylin was used for counterstaining. Hypoxic area fractions were determined by image analysis.
Anti α sma rabbit polyclonal antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-α-SMA rabbit polyclonal antibody is a laboratory reagent used for the detection and analysis of alpha-smooth muscle actin (α-SMA) in various biological samples. It is a rabbit-derived polyclonal antibody that specifically recognizes and binds to α-SMA, a cytoskeletal protein commonly used as a marker for smooth muscle cells and myofibroblasts.
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Lab products found in correlation
Anti collagen 1 rabbit polyclonal antibody, by Abcam (1 mentions)
Anti vimentin rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by GE Healthcare (1 mentions)
Polyvinylidene fluoride membranes, by Cell Signaling Technology (1 mentions)
Anti e cadherin rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Anti fibronectin mouse monoclonal antibody, by BD (1 mentions)
Anti phospho smad2 antibody, by Cell Signaling Technology (1 mentions)
Anti n cadherin rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Goat anti rabbit horseradish peroxidase labeled antibody, by Abcam (1 mentions)
3 3 diaminobenzidine dako, by Agilent Technologies (1 mentions)
Slidebook 4, by Olympus (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Fugene hd, by Promega (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Nigericin, by Merck Group (1 mentions)
Tak 242, by Merck Group (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Poly da dt, by Merck Group (1 mentions)
5 protocols using anti α sma rabbit polyclonal antibody
1
Tumor Hypoxia and Pericyte Marker Analysis
2
Western Blot Analysis of Cellular Signaling
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Cells were lysed with Nonidet P-40 (NP-40) lysis buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% NP-40) containing protease inhibitor cocktail. Proteins were separated on sodium dodecyl sulfate‑polyacrylamide gel electrophoresis gels and were electroblotted onto polyvinylidene fluoride membrane (GE Healthcare Bio-sciences, Piscataway, NJ). The polyvinylidene fluoride membranes were incubated with anti-phospho-Smad2 antibody (Ser465/467), anti-phospho-extracellular-signal-regulated kinases (ERK) 1/2 monoclonal antibody (Thr202/Tyr204), anti-total-Smad2 mouse monoclonal antibody, anti-total-ERK1/2 rabbit monoclonal antibody, anti-E-cadherin rabbit monoclonal antibody, anti-vimentin rabbit monoclonal antibody, anti-N-cadherin rabbit monoclonal antibody (Cell Signaling, Danvers, MA), anti-α-SMA rabbit polyclonal antibody, anti-collagen I rabbit polyclonal antibody (Abcam), or anti-fibronectin mouse monoclonal antibody (BD Biosciences). The HRP‑conjugated goat anti-rabbit or anti-mouse IgG (GE Healthcare Bio-sciences) served as the secondary antibodies. The immunolabeled proteins were visualized by enhanced chemiluminescence. The band intensity was analyzed densitometrically by using ImageJ software (NIH).
3
Histological Analysis of Cellular Spheroids
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The histological analysis was performed as per a previously described protocol. In brief, harvested spheroids were fixed in 4% paraformaldehyde for 15 min at room temperature, followed by sectioning. For morphological analysis, spheroids sections were stained with hematoxylin and eosin (H&E) (18 (link)). Furthermore, α-SMA staining in spheroids was performed as per a previous protocol (19 (link)). Immunohistochemical staining was performed using anti α-SMA rabbit polyclonal antibody (cat no. Ab5694; 1:200 dilution; Abcam) and horseradish peroxidase labeled goat anti-Rabbit antibody (cat. no. AB_2630375; Dako; Agilent Technologies, Inc.) along with 3,3-diaminobenzidine (Dako; Agilent Technologies, Inc.).
4
Immunofluorescence Staining of Mouse Lung Fibroblasts
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Mouse lung fibroblasts were cultured to sub-confluence on glass slides covered with 1% gelatin in DMEM containing 10% FBS, after which cells were washed with cold PBS, fixed in methanol at −20 °C for 4 min, and washed with cold PBS twice, followed by incubation with anti-α-SMA rabbit polyclonal antibody from Abcam (Cambridge, MA, USA) (1:500) for 1 h at room temperature. Cells were washed three times with cold PBS, incubated with Alexa Fluor 488 anti-rabbit IgG (1:200) and DAPI (1:10,000) for 1 h at room temperature, then washed with cold PBS, air-dried, covered, and sealed. Images were acquired with an Olympus IX71 fluorescence microscope (Olympus, Tokyo, Japan) equipped with objective x60/1.42 and Olympus Slidebook 4.1 software.
5
Inflammasome Activation Protocol
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JT001 was synthesized at Jecure Therapeutics (San Diego, CA, USA). Emricasan was purchased from Selleckchem (Houston, TX, USA) and TAK-242 was purchased from Sigma-Aldrich (St. Louis, MO. USA). Nigericin, lipopolysaccharide (LPS) E. Coli O26:B6, Poly(dA:dT) and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Flagellin (FLA-BS Ultrapure), MSU, L18-MDP muramyl-dipeptide, and PAM3CSK4 were purchased from Invivogen (San Diego, CA, USA). FuGENE HD and CellTiter-Glo luminescent cell viability assay were purchased from Promega (Madison, WI, USA). The following primary antibodies were used: anti-ASC mouse monoclonal antibody (clone 2EI-7, Millipore-Sigma, St.
Louis, MO, USA), anti-MPO rabbit polyclonal antibody (ThermoFisher, Waltham, MA, USA), anti-procaspase-1 + p10 + p12 rabbit monoclonal (clone EPR16883, Abcam, Cambridge, UK), anti-αSMA rabbit polyclonal antibody (clone EPR5368, Abcam, Cambridge, UK), anti-NLRP3 mouse monoclonal (clone Cryo-2, Adipogen, San Diego, CA, USA) and anti-β-actin mouse monoclonal (clone AC-74, Millipore-Sigma, St. Louis, MO, USA). Secondary antibodies included goat anti-mouse IgG Alexa Fluor-488 (Life Technologies, Carlsbad, CA, USA) and horse-radish peroxidase-conjugated secondary antibodies (Azure Biosystems, Dublin, CA, USA).
Louis, MO, USA), anti-MPO rabbit polyclonal antibody (ThermoFisher, Waltham, MA, USA), anti-procaspase-1 + p10 + p12 rabbit monoclonal (clone EPR16883, Abcam, Cambridge, UK), anti-αSMA rabbit polyclonal antibody (clone EPR5368, Abcam, Cambridge, UK), anti-NLRP3 mouse monoclonal (clone Cryo-2, Adipogen, San Diego, CA, USA) and anti-β-actin mouse monoclonal (clone AC-74, Millipore-Sigma, St. Louis, MO, USA). Secondary antibodies included goat anti-mouse IgG Alexa Fluor-488 (Life Technologies, Carlsbad, CA, USA) and horse-radish peroxidase-conjugated secondary antibodies (Azure Biosystems, Dublin, CA, USA).
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