CFs were washed with PBS (0.1 mol/L, pH 7-7.4) three times, lysed in a lysis buffer for 30 min at 4 °C, and then scraped off using a cell scraper before being mixed with a one-quarter volume of 5× loading buffer. The cell protein mixture was then boiled in a 100 °C water bath and cooled to room temperature. The protein concentration was determined using a Bradford protein assay kit (Amresco, M173-KIT). Equal amounts of protein (50 µg) were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The strips were blocked with 5% nonfat milk for 1 h at room temperature. The strips were then incubated overnight with primary antibodies against AAT1 (Sigma, AV48205, 1:500), collagen I (Abcam, ab254113, 1:500), collagen III (Abcam, ab7778, 1:500), α-SMA (Abcam, ab5694, 1:500), β-catenin (Abcam, ab6302, 1:500), p-P38 MAPK (Abcam, ab4822, 1:500), P38 MAPK (Abcam, ab170099, 1:500) and GAPDH (Abcam, ab181602, 1:2000) diluted in PBS with 0.05% Tween 20 at 4 °C. The strips were rinsed using PBS with 0.05% Tween 20 for 30 min, followed by incubation with secondary antibodies for 1 h at room temperature. All protein bands were detected using Amersham ECL Western blotting detection reagents (GE Healthcare, RPN2106) and analyzed with a biological electrophoresis image analysis system.
Ab254113
Manufactured by Abcam
Sourced in United States
Ab254113 is a product offered by Abcam. It is a laboratory equipment item, but a detailed factual description is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab175186, by Abcam (2 mentions)
Ab6276, by Abcam (2 mentions)
Ab6302, by Abcam (1 mentions)
Ab4822, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab7778, by Abcam (1 mentions)
Bradford protein assay kit, by Avantor (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab170099, by Abcam (1 mentions)
Ab8069, by Abcam (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab2413, by Abcam (1 mentions)
3 3 diaminobenzine dab, by Thermo Fisher Scientific (1 mentions)
Tris edta buffer, by Boster Bio (1 mentions)
Sv0004, by Boster Bio (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
Zli 9017, by ZSGB-BIO (1 mentions)
12 protocols using ab254113
1
Protein Expression Analysis of Cellular Extracts
2
Histological Analysis of Urethral Tissue
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Four weeks after surgery, the urethral tissue was excised and fixed in 4% paraformaldehyde overnight, followed by routine histological procedures. The sections (4 μm) were stained with hematoxylin and eosin (H&E), Masson's trichrome (MTS), and immunofluorescence (IF) to evaluate the morphology and fibrosis of the urethral tissue. IF was performed using anti-α-SMA antibody (1:200, ab7817, Abcam), anti-vimentin antibody (1:200, ab8069, Abcam), anti-COL1 antibody (1:200, ab254113, Abcam), and anti-CD31 antibody (1:200, 3528, CST) according to the instructions.
3
Immunohistochemical Analysis of Rat Kidney
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Paraffin-embedded rat kidney slides were deparaffined, rehydrated, and immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. All sections were heated in Tris-EDTA buffer (pH 9.0, Boster, Wuhan, China), blocked with 5% blocking buffer for 30min at 37°C, incubated with primary antibodies against fibronectin (FN) (1:100, Abcam Cat# ab2413, Cambridge, England), type I collagen (Col-I) (1:100, Abcam Cat# ab254113, Cambridge, England) at 4°C overnight, incubated with species-specific secondary antibody (SV0004, Boster, Wuhan, China), developed with 3,3′-diaminobenzine (DAB, Invitrogen, California, United States) and counterstained with hematoxylin. The integrated optical density (iod) values of the positive staining areas were measured by ImagePro Plus 6.0 software (Media Cybernetics, CA, United States).
4
Immunohistochemical Analysis of Foot Tendons
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All the foot tendon tissue sections were fixed in 4% formalin overnight and embedded in paraffin with standard techniques. After washing with PBS, the sections were incubated with primary antibodies overnight at 4°C as following: anti-LC3 antibody (1:200, ab48394, Abcam) and anti-Collagen 1 antibody (1:200, ab254113, Abcam). After washing three times in PBS, the sections were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:5000; ab205718, Abcam) for 30 min at 37°C and stained with DAB and Hematoxylin. The stained slides were recorded using a Nikon D300 microscope (Nikon, Tokyo, Japan).
5
Immunohistochemical Analysis of Collagen I
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Paraffin tissue sections were incubated at 65 °C for 20 min then treated with dewax solution using concentration gradients of xylene and alcohol. After washing with PBS, tissue sections were treated with citrate buffer (pH 6.0), they were exposed to heat-induced epitope retrieval (100 °C) for 3 min and heating for 20 min. After treatment with 3% hydrogen peroxide for 10 min and goat serum at 37 °C for 30 min, the tissue sections were incubated with primary antibodies against anti-Collagen I (Abcam, ab254113, 1:200) at 4 °C overnight. The tissue sections were subsequently incubated with secondary antibodies (Goat anti-Rabbit, PV-6001) at room temperature for 60 min. After washing with PBS, these sections were stained using 3,3-diaminobenzidine (DAB, ZSGB-BIO, ZLI9017). The staining was terminated when the tissue sections were brown. Subsequently, all the tissue sections were counterstained with hematoxylin for 12 s. Finally, these sections were dehydrated with ethanol and toluene then sealed with neutral gum. Photographs were taken under microscope and the percentage of positive area was taken as statistical basis.
6
Immunohistochemical Localization of COLI
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Cell slides were quickly placed in cold acetone for 30 min. After soaking in the permeabilizing solution for 5 min, wash with distilled water. 50 μL of 1 : 20 diluted goat serum was added dropwise and incubated at 37 °C for 15 min. After aspirating the serum, 1 : 200 dilution of COLI (ab254113, Abcam, USA) antibody was added and incubated overnight at 4 °C. After washing with PBS on a shaker, IgG H&L (HRP) secondary antibody (ab205718) was added dropwise and incubated at 37 °C for 40 min. DAB (25 mg/ml) was used for color developed and then washed with tap water. Cell slides were dehydrated with graded alcohol (80% for 2 min, 95% for 2 min, and 100% for 5 min × 2). After transparent treatment with xylene, the slides were sealed with neutral gum and observed under a microscope (× 400).
7
Ginsenoside Rb1 Attenuates Hepatic Stellate Cell Activation
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Primary antibodies against alpha-smooth muscle actin (α-SMA; ab124964), Type I collagen (Col1A1; ab254113 and ab138492), β-actin (ab6276), Beclin 1 (BECN1; ab207612), and SLC7A11 (ab175186) were obtained from Abcam (Shanghai, China). GRb1 (≥95% purity; Y0001347), CCl4 (488488), curcumin (Cur; C1386 ), ferrostatin-1 (Fer-1; SML0583 ), Z-VAD-FMK (219007 ), necrostatin-1 (Nec-1; 480065 ), and erastin (Era; 329600 ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The chemical structure of GRb1 is shown in Fig. 1 A.Fig. 1 ![]()
Ginsenoside Rb1 (GRb1) suppresses hepatic stellate cell (HSC) activation in LX-2 cells. LX-2 cells were treated with varying concentrations of GRb1 (0, 2.5, 5, 10, 20, and 40 μM) over 24 h. (A) Representation of GRb1's chemical structure. (B) Cell viability post GRb1 treatment. (C, D) Messenger RNA (mRNA) expression levels of alpha-smooth muscle actin (α-SMA) (C) and type I collagen (Col1A1) (D). (E) Protein expression levels of α-SMA and Col1A1. (F) Co-localization of α-SMA (shown in red) and Col1A1 (shown in green) in LX-2 cells was analyzed using immunofluorescence staining. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei in blue. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Cur: curcumin.
8
Bladder Tissue Protein Extraction and Western Blot
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After cutting the bladder tissue into pieces with scissors, it was placed in a homogenizer. A mixture of NP40 lysate was added (Beyotime, P0012F, China), then 1% protease inhibitor (Roche, 4693159001, Switzerland), and was homogenized repeatedly until there was no obvious precipitation. The tissue was then centrifuged at 12,000 rpm at 4 °C for 5 min, with the supernatant being extracted. The protein concentration was determined by a BCA reagent test kit (Beyotime, P0010, China). The protein sample (30 µg) was separated by SDS-PAGE gel and transferred to a PVDF membrane and blocked for 1 hour, incubated with collagen I (Abcam, ab254113, America, rabbit, 1:1,000), mast cell protease 1 (Affinity, DF12290, China, rabbit, 1:1,000), AOAH (Affinity, DF9158, China, rabbit:cat 1:1,000), GAPDH (Abcam, ab8245, America, mouse, 1:5,000), and β- actine (Abcam, ab6276, America, mouse, 1:5,000) overnight at 4 °C. Membranes were labeled with the appropriate secondary antibody and visualized by enhanced chemiluminescence. Quantitative analysis of blots was performed by densitometry using Image J software.
9
Protein Expression Analysis of Heart Tissues
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The total proteins were extracted from heart tissues, or cells by using ice-cold RIPA lysis buffer (Beyotime, China). A total of 30 μg proteins were separated by SDS-polyacrylamide gel (SDS-PAGE), shifted to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany), and blocked in 5% non-fat milk, followed by incubation with specific primary anti-collagen 1 (ab254113, Abcam, United States, 1:1,000), anti-α-SMA (ab40854, Abcam, United States, 1:1,000), anti-GPX4 (ab252833, Abcam, United States, 1:1,000), anti-SLC7A11 (ab175186, Abcam, United States, 1:1,000) antibodies at 4°C overnight. Next day, the protein bands were incubated with HRP conjugated secondary anti-mouse or anti-rabbit antibodies (ab175775/ab288151, Abcam, United States, 1:1,000) at room temperature for 1 h. The bands were then visualized by using ECL (Thermo) on a gel image system (Bio-Rad, United States). All antibodies were purchased from Abcam.
10
Histological Assessment of Rat Kidney
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The cortex of the right kidneys of rats was dissected and at −80°C for RNA protein extraction. The remaining kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm sections. Then, the sections were stained with a Periodic Acid-Schiff (PAS) staining kit (Sbjbio Life Sciences, Nanjing, Jiangsu, China) and observed under an optical microscope (magnification × 400). The proportion of PAS staining area to total area of the glomerulus was examined using the Image J software (NIH). In each group of kidney tissues, 20 glomeruli were collected. In addition, hematoxylin and eosin (HE) staining was conducted to examine the pathological changes in rat kidney tissues. The staining in 20 random glomerular areas was scored by three pathologists who had no idea of the grouping details, and the scoring was performed according to tubular cell necrosis, cytoplasmic vacuole formation, hemorrhage, and tubular dilatation.15 (link) Moreover, immunohistochemical staining was performed to examine the protein level of a fibrosis-marker collagen I (Col. I) in the rat kidney tissues using anti-collagen I (1:200, ab254113, Abcam). To each tissue section, 20 continuous fields of views were observed and the integrated optical density (IOD) value was examined using the Image J to examine the expression of Col. I.
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