Immunofluorescence staining was performed as described before [16 (link)] with some modification, to detect the expression levels and differences of fibronectin (FN), collagen I (Col I), and α-smooth muscle actin (α-SMA) in the kidneys of five groups (Mod group, Mang-H group, BpV group, and BpV+Mang-H group). The sections of the paraffin-embedded sample were made and blocked with serum-free protein (Dako, Victoria, Australia) and permeabilized for 30 min. Sections were incubated with primary antibodies FN, Col I, α-SMA (Wanleibio, Shijiazhuang, China; 1 : 100 dilution) overnight at 4°C. Sections were washed with TBS 3× 10 min and incubated with species-specific secondary antibodies: Goat Anti-Rabbit IgG H&L (Cy3®) (1 : 1000 dilution, ab6939, Abcam, USA) at room temperature for 1 h. After washing with TBS, the sections were stained with Glycerol Mounting Medium (Abcam) that contained 4,6-diamidino-2-phenylindole (DAPI) and 1,4-diazobicyclo-2,2,2-octane (DABCO). Labelled tissues were visualized using a Leica DM LB2 microscope. Fluorescence images (400x magnification) were captured using NIS-Elements 4.13 (Nikon, Japan) software. The number of fluorescence-positive cells was counted from five representative high-power fields (400x magnification) per tissue section.
α sma
Manufactured by Wanlei
Sourced in China
α-SMA is a cytoskeletal protein that is commonly used as a marker for smooth muscle cells. It is involved in the contractile function of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Dm lb2 microscope, by Leica (1 mentions)
Nis elements 4, by Nikon (1 mentions)
Goat anti rabbit igg h l cy3, by Abcam (1 mentions)
E cadherin, by Wanlei (1 mentions)
Tnf α, by Wanlei (1 mentions)
Sp kit sp 9000, by ZSGB-BIO (1 mentions)
Il 1β, by Wanlei (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ccnd2, by Proteintech (1 mentions)
β tubulin, by Proteintech (1 mentions)
P erk1 2, by Wanlei (1 mentions)
Ki 67, by Wanlei (1 mentions)
Bradford method, by Solarbio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
P jnk, by Wanlei (1 mentions)
Horseradish peroxidase conjugate secondary antibody, by Santa Cruz Biotechnology (1 mentions)
6 protocols using α sma
1
Quantitative Immunofluorescence Assay for Renal Fibrosis
2
Investigating MXD's Inhibition of TGF-β/Smad3 Pathway
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To elucidate whether the protection of MXD was mediated by the inhibition of the TGF-β/Smad3 signal pathway, a verification experiment was conducted using rTGF-β (Neobioscience, Shanghai, China). Briefly, cells were grouped as follows: (1) control group; (2) PM2.5 group; (3) treatment group, cells challenged by PM2.5 exposure were incubated with 20% MXD-medicated serum; and (4) rTGF-β group, cells undergone PM2.5 stimulation were incubated with 20% MXD-medicated serum + rTGF-β (10 ng/mL). Medicated serum (or nonimmune serum) and rTGF-β were added simultaneously right after the stimulation of PM2.5. After a 48-hour incubation, cells were photographed and harvested. The expression of E-cadherin, α-SMA (Wanleibio, Shenyang, China; dilution 1 : 1000), vimentin, p-Smad3, ZO-1, and claudin-5 was semiquantitatively analysed.
3
Immunohistochemical Analysis of Lung Fibrosis
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Briefly, endogenous peroxidase activity within the sections was quenched by incubating the sections with 3% H2O2 for 10 min after dewaxing and hydration. Lung tissues were incubated in a humidified chamber with primary antibodies directed against Collagen I (1:100; cat. no. AF7001; Affbiotech), fibronectin (1:100; cat. no. WL00712a; Wanleibio) and α-SMA (1:100; cat. no. WL02510; Wanleibio) overnight at 4°C. On the following day, the lung tissues were washed with PBS and incubated with a IgG antibody (1:100; cat. no. ab150077; Abcam) at 37°C for 45 min. In the negative controls, the primary antibody was replaced with PBS. Subsequently, tissues were counterstained with DAB.
4
Immunohistochemical Analysis of Skin and Liver Samples
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The sections from the above paraffin‐embedded skin and liver samples were also deparaffinized and subjected to IHC staining as described.45 , 48 , 51 , 52 Specifically, the sections were deparaffinized and rehydrated. After antigen retrieval, the sections were subjected to immunostaining with antibodies against CD45 (1:100 dilution; Wanleibio; Cat# WL00922), CD54 (1:100 dilution; Wanleibio; Cat# WL02268), CD40L (1:50 dilution; Bimake; Cat. No. A5778), CD3D (1:50 dilution; Bimake; Cat. No. A5886), TNFα (1:200 dilution; Wanleibio; Cat# WL01896), IL1β (1:200 dilution; Wanleibio; Cat# WL00891), CD20 (1:200 dilution; Wanleibio; Cat# WL02883), IL10 (1:200 dilution; Wanleibio; Cat# WL03088), Collagen I (1:100 dilution; Wanleibio; Cat# WL0088), α‐SMA (1:50 dilution; Wanleibio; Cat# WL02510), or TIMP1 (1:100 dilution; Wanleibio; Cat# WL02342). The proteins of interest were detected with the biotin labeled goat anti‐rabbit IgG or anti‐mouse IgG/streptavidin‐HRP kit (SP Kit, SP‐9000, ZSGB‐Bio). Minus primary antibody and/or rabbit IgG and mouse IgG were used as negative controls. Staining results were recorded under a bright field microscope (Leica, DM4B).
5
Western Blot Analysis of Protein Expression
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Proteins were extracted using RIPA buffer (Solarbio, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride (Solarbio). Protein concentration was determined using the Bradford method (Solarbio). Equal amounts of proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Merck, Germany). After blocking with 5% non-fat milk in TBST for 2 h, the membranes were incubated with primary antibodies against CCND2 (1:500, Proteintech, Wuhan, China), PCNA (1:1,000, Proteintech), Ki-67 (1:500, Wanleibio, Shenyang, China), p-ERK1/2 (1:300, Wanleibio), p-p38 (1:750, Wanleibio), p-JNK (1:300, Wanleibio), MMP9 (1:1,000, Wanleibio), MMP2 (1:500, Wanleibio), α-SMA (1:500, Wanleibio), GAPDH (1:1,000, Proteintech) and β-tubulin (1:1,000, Proteintech) at 4°C overnight. After washing with TBST, the membranes were then incubated with goat anti-rabbit IgG secondary antibody (dilution at a 1:20,000, Wanleibio) at 37°C for 2 h. Signal was visualized by ChemiDocTM MP Imaging System (BIO-RAD, California, USA). The experiment was repeated at least three times.
6
Evaluating p10/OA Complex Effects on Cell Markers
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HSC-T6 cells were plated in a six-well plate at a density of 5.0 × 10 5 cells per well and cultured overnight in an incubator. For 24 h, cells were treated with various molar ratios (8:16 and 8:20) of preformed p10/OA complex. After three times washing with cold PBS, cells were lysed using RIPA lysis buffer containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Equal quantities of protein (approximately 10-20 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% nonfat milk in TBST for 1 h, the PVDF membrane was incubated overnight at 4 °C with rabbit polyclonal α-SMA (Wanleibio; 1:1000) and mouse monoclonal COL1α2 primary antibody (Servicebio; 1:1000). Following TBST washes 3 times, the PVDF membrane was incubated for 1 h at room temperature with horseradish peroxidase conjugate secondary antibody (Santa Cruz biotechnology; 1:3000). The Clinx ChemiScope 3000 mini enhanced chemiluminescence (ECL) detection reagent was used to detect a chemical reaction light signal.
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