Cultured porcine SMCs were washed twice with phosphate-buffered saline (PBS; Corning, New York, NY, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Subsequent to incubation with primary (anti-Cx40, anti-Cx43, anti-S100A4 and anti-α-smooth muscle actin (SMA); Abcam, Cambridge, MA, USA) and polymer helper and poly-peroxidase anti-mouse/rabbit immunoglobulin G secondary antibodies (PV-9000 kit; GBI Labs, Mukilteo, WA USA) were added for 1 h, and diaminobenzidine (Roche Diagnostics GmbH) was used for signal detection. The slides were washed under running water and tissue samples were counterstained with hematoxylin. Sections were observed and imaged using an Olympus CX31 microscope (Olympus Corporation, Tokyo, Japan).
Anti α smooth muscle actin
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-α-smooth muscle actin is a protein that is commonly used as a marker for smooth muscle cells. It is a structural protein that plays a role in the contraction of smooth muscle cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd31, by Abcam (4 mentions)
Anti vimentin, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Anti cx43, by Abcam (1 mentions)
Anti s100a4, by Abcam (1 mentions)
Anti cx40, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Cx31 microscope, by Olympus (1 mentions)
Anti cav 1, by Santa Cruz Biotechnology (1 mentions)
Anti cd31, by Santa Cruz Biotechnology (1 mentions)
Anti pedf, by Merck Group (1 mentions)
Anti cav 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti il 33, by R&D Systems (1 mentions)
Anti tslp, by Abcam (1 mentions)
Anti wheat germ agglutinin, by Abcam (1 mentions)
Anti cyclophilin a, by Abcam (1 mentions)
Anti occludin, by Abcam (1 mentions)
22 protocols using anti α smooth muscle actin
1
Immunohistochemical Analysis of Porcine SMCs
2
Analyzing Human and Mouse Skin Biopsies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry (IHC) analysis of human skin biopsies was performed on 3 µm paraffin sections using mouse monoclonal anti-PEDF (Clone 10F12.2; Millipore, UK), rabbit polyclonal anti-CD31, rabbit polyclonal anti-α-smooth muscle actin (SMA) (Abcam, UK) and a rabbit polyclonal anti-Cav-1 antibody (Santa Cruz, UK). Detailed procedure of the two-step staining is described in the online supplementary methods . The number of positive cells was counted by two pathologists, blinded to tissue source and expressed as the mean of two observations for each sample. For mouse skin biopsies, we employed rabbit polyclonal anti-PEDF (Aviva Systems Biology; Insight Biotech, UK), rabbit polyclonal anti-CD31 (Santa Cruz) and rabbit polyclonal anti-Cav-1 (Santa Cruz) antibodies. All sections were imaged using an Axioplan Zeiss light microscope equipped with an AxioCam digital camera.
3
Quantification of Lung Inflammation Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung sections were incubated with the following primary antibodies: anti-α-smooth muscle actin (SMA, 1:200; Abcam, Cambridge, UK), anti-IL-33 (1:50; R&D Systems, Minneapolis, MN, USA), anti-TSLP (1:4,000; Abcam), and anti-IL-23R (1:200; Abcam). For isotype controls, anti-rabbit or anti-goat IgG antibodies were used. IHC staining was photographed using a Nikon light microscope and analyzed with digital imaging software (iSolution Lite, IMT i-Solution Inc., Daejeon, Korea). Slides were examined at ×400 magnification. The average percent area of IHC stainings was quantified as positive areas with 4–5 fields/mouse (n = 3) using ImageJ software (NIH, Bethesda, MD, USA) after setting the thresholds.
4
Cardiac Fibrosis and Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heart tissues were post-fixed overnight in 4% paraformaldehyde, then embedded in paraffin on the embedding station. Heart sections (5 μm) were stained with trichrome for the assessment of cardiac fibrosis and analyzed using ImagePro Plus version 6.0. For immunofluorescence, heart sections were stained with anti–α-myosin heavy chain (MHC) (ABclonal, Wuhan, China), anti–β-MHC (ABclonal, Wuhan, China), anti–wheat germ agglutinin (WGA) (Abcam, Cambridge, UK), anti-collagen Iα (Abcam, Cambridge, UK) and anti–α-smooth muscle actin (SMA) (Abcam, Cambridge).
5
Immunocytochemical and Flow Cytometric Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used: rabbit pAb, IgG, anti–von Willebrand factor (vWF) at 1:100 for immunocytochemistry (ICC); 1:20 for flow cytometry (FC; Abcam, Cambridge, MA, USA), mouse mAb, IgG1, anti–platelet endothelial cell adhesion molecule-1 (PECAM-1, 1:200; 1:20; Abcam), rabbit mAb, IgG, anti-β catenin (1:250; 1:10) (Abcam), rabbit pAb, IgG, anti–α-smooth muscle actin (SMA, 1:100; 1:20; Abcam), rabbit pAb, IgG, anti–glial fibrillary acidic protein (GFAP; 1:200; 1:25), and mouse mAb, IgG, anti-vimentin (1:500; 1:25; Agilent Technologies) and mouse mAb, IgG, anti-chondroitin sulfate (NG2, 1:500; 1:25; R&D Systems). Rabbit pAb, IgG, anti-occludin (1:200; Abcam) was used for ICC and Western blot (WB). Rabbit pAb, IgG, and anti-cyclophilin A (Abcam) were used as a loading control for WB at 1:10,000.
6
Immunofluorescent Staining of Cardiac Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen sections of heart tissues were prepared by fixing with OCT (cat. no. 4583; Sakura Finetek USA, Inc.) and sectioned at 4 weeks after MI. Slides were permeabilized by 0.1% Triton for 10 min, blocked in 1% bovine serum albumin, and then incubated with following primary antibodies: anti-Von Willebrand factor (vWF; cat. no. ab6994; Abcam, Cambridge, UK), anti-α-smooth muscle actin (SMA; cat. no. ab5694; Abcam), and anti-vimentin (cat. no. ab8978; Abcam). After rinsing with PBS, the cells were incubated in PE-conjugated goat anti-mouse IgG secondary antibodies (cat. no. A10703G-PE; Solarbio) or Cy3-conjugated goat anti-rabbit IgG (cat. no. EK022; Zhuangzhibio, China). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).
7
Immunofluorescence Staining of Tumor and Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors and thigh muscles were cryopreserved and embedded in optimal cutting temperature compound (Tissue‐Tek, SAKURA Finetek USA, Torrance, CA) for immunofluorescence staining. Five‐µm sections were incubated with anti‐CD31 (Abcam, Cambridge, UK) and anti‐α‐smooth muscle actin (SMA; Abcam) antibodies to detect endotheliocytes and pericytes, respectively. Nuclei were stained with DAPI (Sigma‐Aldrich, St. Louis, MO). A pericyte‐positive microvessel was defined as a CD31‐positive microvessel surrounded by at least one cell staining positive for α‐SMA.
For the ultrastructural observations, specimens were prepared as described elsewhere26 and slides were visualized under a transmission electron microscope (JEM‐2010HR, JEOL, Tokyo, Japan).
For the ultrastructural observations, specimens were prepared as described elsewhere
8
Molecular Mechanisms of Hepatic Stellate Cell Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human hepatic stellate cell line (LX-2) and the human embryonic kidney cell line cells (HEK 293T) were obtained from American Type Culture Collection (ATCC). Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS) and Opti-MEM were obtained from GIBCO (Los Angeles, CA, USA). Anti-Akt, anti-phospho-Akt, anti-GAPDH antibodies, and LY 294002 (PI3K inhibitor) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-FOG2 and anti-α-smooth muscle actin (SMA) antibodies were purchased from Abcam (New Territories, Hong Kong). Anti-vimentin antibody was obtained from Millipore (Schwalbach am Taunus, Germany). Lipofectamine 2000 and Trizol were obtained from Invitrogen. Cell Counting Kit-8 (CCK-8) was from Dojindo (Kumamoto, Japan). miExpress™ Precursor miRNA Expression (pEZX-MR03) clone was ordered from GeneCopoeia (Rockville, MD, USA). Universal-RT-microRNA-PCR kits were from EXIQON (Vedbaek, Denmark). SYBR Green qPCR superMix was from Transgen Biotech (Beijing, China).
9
Protein Expression Analysis in HTFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated from the HTFs using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein was quantified using a BCA protein kit (Thermo Fisher Scientific, Inc.). Proteins were then separated via 10% SDS-PAGE and transferred onto PVDF membranes. Subsequently, the membranes were blocked with 3% non-fat milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Subsequently, membranes were incubated with an anti-rabbit secondary antibody (1:5000) for 1 h at room temperature. Finally, the membranes were visualized with an enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) and the density of blots was analyzed using ImageJ software (National Institutes of Health). The primary antibodies used in the present study were as follows: Anti-α-smooth muscle actin (SMA; Abcam; 1:1000), anti-collagen I (Abcam; 1:1000), anti-survivin (Abcam; 1:1000), anti-autophagy related (ATG)5 (Abcam; 1:1000), anti-Beclin-1 (Abcam; 1:1000), anti-Bax (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control. Moreover, the Western blot raw data are presented in the supplementary material.
10
Quantifying Tumor Vasculature and Hypoxia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse tissue samples were immediately frozen in Tissue-Tek ® O.C.T. compound (SAKURA, Tokyo, Japan) or xed in 10% formalin and prepared in para n. OCT compound-embedded frozen samples were cut into 15-μm thick sections while para n-embedded samples were cut into 3-μm thick sections. Immuno uorescence analysis was performed to evaluate the area densities of the tumor microvessels, vascular normalization, and tumor hypoxia. The frozen tissue sections were xed in 5% paraformaldehyde for 15 min and the para n-embedded tissue sections were depara nized in xylene and hydrated with a graded alcohol series and distilled water. These tissue slides were blocked using 5% goat or donkey serum for 1 h. The sections were then incubated with the following primary antibodies: anti-CD31 (1:200, Angio-Protemie, Boston, MA), anti-α-smooth muscle actin (SMA) (1:100, Abcam, Cambridge, UK), or anti-pimonidazole (1:50, Hypoxyprobe Inc., Burlington, MA) antibody overnight at 4 °C.
The tissues were subsequently washed with phosphate-buffered saline (PBS) and incubated for 1 h at 25°C with a second antibody (diluted 1:400) conjugated to AF488, AF568, or Cy5 (Abcam). The cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The slides were observed under BZ-X800 uorescence (KEYENCE, Osaka, Japan) or laser confocal LSM 780 (Zeiss, Oberkochen, Germany) microscopes.
The tissues were subsequently washed with phosphate-buffered saline (PBS) and incubated for 1 h at 25°C with a second antibody (diluted 1:400) conjugated to AF488, AF568, or Cy5 (Abcam). The cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The slides were observed under BZ-X800 uorescence (KEYENCE, Osaka, Japan) or laser confocal LSM 780 (Zeiss, Oberkochen, Germany) microscopes.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!