The aortic tissues were fixed overnight in 4% formalin, then dehydrated and embedded in paraffin following the conventional method. The paraffin-embedded samples were sectioned into pieces 3 μm thick. The sections were stained with hematoxylin and eosin, trichrome, and Verhoeff-van Gieson staining. For immunohistochemistry, the slides were incubated with alpha smooth muscle actin (αSMA, PA5-85070, ThermoFisher Scientific, Waltham, MA, USA), matrix metalloproteinase-2 (MMP2, sc-13594, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and matrix metalloproteinase-9 (MMP9, ab58803, Abcam, Cambridge, UK). The slides were also examined under a light microscope (Axioplan 2 imaging, ZEISS, Jena, Thuringia, Germany), and the slide images were digitalized and transformed into computerized images.
α sma
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Italy, Japan
α-SMA is a protein marker that is commonly used in immunohistochemistry and western blotting applications to detect the presence of smooth muscle cells. It is a structural protein found in the contractile apparatus of smooth muscle cells.
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Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (9 mentions)
Fibronectin, by Thermo Fisher Scientific (7 mentions)
Vimentin, by Thermo Fisher Scientific (7 mentions)
Collagen 1, by Abcam (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (6 mentions)
β actin, by Thermo Fisher Scientific (5 mentions)
Col1a1, by Thermo Fisher Scientific (5 mentions)
Tgf β, by R&D Systems (4 mentions)
E cadherin, by Thermo Fisher Scientific (4 mentions)
Vectashield mounting medium, by Vector Laboratories (4 mentions)
Desmin, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Thermo Fisher Scientific (3 mentions)
Axioplan 2 imaging, by Zeiss (3 mentions)
P smad2 3, by Santa Cruz Biotechnology (3 mentions)
Prolong gold mounting medium with dapi, by Thermo Fisher Scientific (3 mentions)
67 protocols using α sma
1
Histological Analysis of Aortic Tissues
2
Investigating HSC Activation by HSP70 and GRP78 in Liver Fibrosis
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Rat hepatic stellate cells (HSCs) were isolated as described elsewhere100 (link).
HSCs were stimulated with complete DMEM medium supplemented with HSP70 (15 ng/ml) or GRP78 (3 ng/ml) for 24 h. TLR4 inhibition in HSCs was performed using the same experimental condition employed for primary hepatocytes. HSCs activation was assessed by flow cytometry and real-time PCR by evaluating alpha-smooth muscle Actin (α-SMA) and Collagen type I alpha 1 (COL1A1) protein and gene expression. α-SMA and COL1A1 antibodies were obtained from Thermo Fisher scientific, (Waltham, MA). Antibody for flow cytometry were diluted 1:50 in phosphate buffered saline (PBS). Primer sequences are reported in Supplementary TableS6 . Gating strategy is showed in Supplementary Fig. S17B .
HSCs were stimulated with complete DMEM medium supplemented with HSP70 (15 ng/ml) or GRP78 (3 ng/ml) for 24 h. TLR4 inhibition in HSCs was performed using the same experimental condition employed for primary hepatocytes. HSCs activation was assessed by flow cytometry and real-time PCR by evaluating alpha-smooth muscle Actin (α-SMA) and Collagen type I alpha 1 (COL1A1) protein and gene expression. α-SMA and COL1A1 antibodies were obtained from Thermo Fisher scientific, (Waltham, MA). Antibody for flow cytometry were diluted 1:50 in phosphate buffered saline (PBS). Primer sequences are reported in Supplementary Table
3
Immunohistochemical Analysis of Tissue Fibrosis Markers
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Tissue sections were stained for collagen I (rabbit polyclonal, Abcam, Cambridge, MA, 1:50 dilution), pSMAD-2/3 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, 1:10 dilution), CD163 (mouse clone 10D6, Neomarkers, Fremont, CA, 1:30 dilution), L-TGFβ (goat polyclonal, R&D Systems, Minneapolis, MN, 1:10 dilution), TGFβ (chicken polyclonal, R&D Systems, Minneapolis, MN, 1:10 dilution), and αSMA (mouse clone 1A4, Thermo Fisher, Pittsburgh, PA, 1:100 dilution). Antigen retrieval and staining were done as previously described [5 (link)]. Briefly, formalin-fixed paraffin-embedded tissues samples were deparaffinized in xylene and rehydrated in ethanol. Samples were then placed into a pressure cooker with boiling antigen retrieval buffer (Tris–HCl, EDTA, Tween-20) for 7 min. After allowing for the slides to cool, the sections were blocked with 2 % fetal bovine serum in phosphate-buffered saline for 30 min. Antibodies and fluorescent tags were incubated on each sample for 1 h with washes in between with phosphate-buffered saline with 0.2 % Tween-20. Prolong Gold Mounting Medium with DAPI (Invitrogen) was then applied to the slides, which were then cured in the dark overnight before being imaged.
4
Histological Analysis of Kidney Samples
5
Protein Quantification and Western Blot Analysis
6
Engineered Hydrogel for Vascular Cell Studies
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GelMA with photoinitiator, lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate, was purchased from Intelligent Manufacturing Research Institute (IMRI, GM‐90, Suzhou, China). TNF‐α, ox‐LDL, and IL‐1β were purchased from Sigma‐Aldrich (St. Louis, USA). DAPI, HUVECs, human SMCs, and actin‐tracker green were purchased from Keygen Biotech Co., Ltd. (Nanjing, China). Paraformaldehyde (4% w/v), Triton X‐100, CCK‐8, and phosphate‐buffered saline (PBS) were purchased from Solarbio (Beijing, China). Mouse MCs were purchased from Kemao Biotechnology (Dongguan, China). Penicillin–streptomycin, Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and endothelial basal medium (F‐12K) were purchased from Gibco (Grand Island, USA). Cell labeling kits (Qtracker 525 and 655), α‐SMA, VE‐Cad primary polyclonal antibody, and goat anti‐rabbit IgG (H+L) secondary antibody were obtained from Thermo Fisher (Waltham, USA).
7
Western Blot Analysis of Cellular Proteins
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The whole lysates (30-50) μg of sample protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8-12 % gel, and transferred to polyvinylidene fluoride membranes. After blocking with 5 % skim milk in TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05 % Tween-20), the membranes were immunoblotted with the primary antibodies against CK2α (1:1000), Pfn1 (1:1000), Myosin-X (1:1000), Myosin IIA (1:1000) (NOVUS, Littleton, CT, USA), fibronectin (1:1000), α-SMA (1:1000) (Thermo Fisher Scientific), p-Myosin IIA (1:1000), p-P53 (1:1000), P16 (1:1000) (Cell signaling, Danvers, MA, USA), P21 (1:1000), SMP30 (1:1000), cyclin D1 (1:1000), CDK4 (1:1000), cyclin E (1:1000), CDK2 (1:1000), and β-actin (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then washed, and the primary antibodies were detected by means of goat anti-rabbit IgG (1:10,000) or goat anti-mouse IgG antibodies (1:10,000) (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Thermo Fisher Scientific). Band densitometric quantifications were determined using ImageJ software 1.48v.
8
Quantitative Analysis of Immune Cell Populations in Formalin-Fixed Paraffin-Embedded Tissue
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Formalin-fixed paraffin-embedded tissue sections (4-μm thick) were immunostained with primary antibodies against CD45RO (dilution, 1:200), CD3 (dilution, 1:150), CD8 (dilution, 1:50), forkhead box P3 (FOXP3; dilution, 1:20) and smooth muscle α-actin (α-SMA; dilution, 1:800) (all from Thermo Fisher Scientific, Inc.). 3-Amino-9-ethylcarbazole was used as a chromogen. Negative controls were treated similarly but without the primary antibody. Positively stained cells were evaluated using an Olympus BX-41 microscope (Olympus Corporation, Tokyo, Japan) with an Infinity 2 Lumenera camera (Lumenera Corporation, Ottawa, Canada) and analyzed using commercial Infinity Capture and Infinity Analyze software (version 6.5.2, Lumenera Corporation, Ottawa, Canada). The slides were viewed at a magnification of ×400, and 10 fields were randomly selected for further analysis of antigen expression. Positively stained cells were calculated in each of the 10 representative fields and averaged per group, and the mean cell count score per organ per animal was obtained.
9
Immunohistochemical Analysis of EMT Markers in OSCC
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In order to investigate the incidence of EMT in OSCC, tumor and margin tissues of 6 patients (2 females and 4 males, average age 59.5±9.33, 3 tongue and 3 floor of the mouth tumors) diagnosed with OSCC were obtained at the Clinic of Maxillofacial Surgery of the School of Dental Medicine at the University of Belgrade. The samples were processed by immunostaining. Deparaffinization of 5-µm tissue sections was performed in xylene. The process was repeated two times for 5 min. The sections were processed by hydration with graded ethanol (100, 96, 80, 70, 50%) 2× for 5 min. Pretreatment was performed in 0.1 ml citrate buffer (pH 6.0) for 20 min at 98°C. The samples were incubated in 3% H2O2 for 5 min and rinsed in Tris-buffered saline solution. This was followed by application of the UV blocker for 5 min. The samples were incubated with rabbit polyclonal antibodies for Vimentin, α-SMA, SNAIL and SLUG (Thermo Fisher Scientific) for 20 min. Following rinsing for 5 min, the samples were analyzed with Quatro amplifier for 10 min, Quatro polymer for 10 min, DAB quatro for 5 min and finally counterstained with hematoxylin for 2 min. Between all these phases, the samples were rinsed for 5 min. The images were captured by the Olympus DP70 camera and the Olympus BX50 microscope (Olympus).
10
Western Blot Analysis of Lung Proteins
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The proteins from lung tissues and LFs were extracted using RIPA buffer containing protease/phosphatase inhibitors (Thermo Fisher Scientific). BCA Protein Assay Kit (Beyotime Biotechnology) was used to quantify the concentration of proteins. After boiling for 5 minutes at 95°C, sample proteins (25 μg) were separated using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene difluoride membranes (PVDF; Merck Millipore). The membranes were blocked with 5% skim milk for 1 hour at room temperature and then incubated with the primary antibodies against TLR9 (1:1000, Abcam), Smad2 (1:500, Abcam), α‐SMA (1:500, Thermo Fisher Scientific), CCN2 (1:1000, Abcam), p‐p38 (1:2000, Thermo Fisher Scientific), p38 (1:1000, Abcam), p‐AKT (1:1000, Thermo Fisher Scientific), AKT (1:500, Abcam), p‐p65 (1:1000, Cell Signaling Technology), p65 (1:500, Abcam), β‐actin (1:500, Abcam) or GAPDH (1:500, Abcam) overnight at 4°C. Then, the membranes were incubated with the horseradish peroxidase‐conjugated secondary antibodies (1:1000, Abcam) for 2 hours at room temperature. Blots were visualized using the ECL Western Blotting Substrate (Thermo Scientific) on ChemiDoc MP Imaging System (Bio‐Rad).
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