The paraffin sections were dewaxed, rehydrated, and incubated with 3% H2O2 to remove endogenous peroxidase. Then, sections were added citrate (10 mM, pH 6.0) buffer for antigen repair and sealed with 10% goat serum for 1 h at 37°C. The liver sections were incubated with primary antibodies Transforming Growth Factor-β (TGF-β, Bioss, bs-0086 R) and Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4, Affinity, DF12141) for one night at 4°C. Then, the sections were incubated with the HRP-conjugated second antibody for 1 h at 37°C. Finally, sections were stained with a DAB kit and sealed with a mounting medium. The mean density of positive protein was calculated by IPP 6.0 software.
Bs 0086r
Manufactured by Bioss Antibodies
Sourced in China
The Bs-0086R is a piece of lab equipment manufactured by Bioss Antibodies. It is designed for general laboratory use. The core function of the Bs-0086R is to provide a reliable and consistent measurement or analysis tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Ab179483, by Abcam (2 mentions)
Bs 0743r, by Bioss Antibodies (2 mentions)
Tgf β, by Bioss Antibodies (1 mentions)
Acsl4, by Affinity Biosciences (1 mentions)
Bs 6761r, by Bioss Antibodies (1 mentions)
Ba400 microscope, by Motic (1 mentions)
Bs1665r, by Bioss Antibodies (1 mentions)
Bs5085r, by Bioss Antibodies (1 mentions)
Bs1007r, by Bioss Antibodies (1 mentions)
Horseradish peroxidase labeled goat rabbit igg h l, by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab171941, by Abcam (1 mentions)
Ab124937, by Abcam (1 mentions)
Iseq00010, by Merck Group (1 mentions)
R0010, by Solarbio (1 mentions)
Cw0102, by CWBIO (1 mentions)
Cw0156, by CWBIO (1 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
T6199, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
11 protocols using bs 0086r
1
Immunohistochemical Analysis of TGF-β and ACSL4
2
Immunohistochemical Analysis of Tumor Markers
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Tissues were fixed in 10% buffered formalin and embedded in paraffin. Paraffin sections of tumor tissues were prepared, dewaxed by dimethyl-benzene, and hydrated with different concentrations of ethanol (100%, 95%, 85%, 70%, and 50%). The following steps were performed: blocking of endogenous peroxidase activity in 3% H2O2 solution, unmasking of the antigenic epitope with citrate buffer, incubation with blocking buffer for blocking, and incubation with primary antibody (VEGF (Bioss, BS1665R), CD34 (Bioss, BS5085R), TGF-β1(Bioss, BS0086R), IL-10 (Bioss, BS6761R), and EGFR (Bioss, BS1007R)) and secondary antibody (horseradish peroxidase-labeled goat rabbit IgG (H + L) (Beyotime, A0208)). Lastly, the DAB (Beyotime) substrate solution was applied to reveal the color of antibody staining. Images were acquired by an Motic BA400 microscope (Motic, China). The expression of cytokines was analyzed by Image-Pro Plus 6.0 (Media Cybernetics, USA).
3
Protein Expression Analysis in Muscle Cells
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Frozen GAS and C2C12 cells were homogenized in RIPA buffer (R0010, Solarbio) containing 1 mM PMSF (P0010, Solarbio), and protease inhibitor cocktail (539133, Merck, Rahway, NJ, USA). The protein concentration was measured using a BCA protein assay kit (P0012, Beyotime, Shanghai, China). Equivalent proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride (PVDF) membranes (ISEQ00010, Merck). After blocking the membrane with 5% non-fat dry milk for 1.5 h, primary antibodies were proceeded overnight at 4 °C and included rabbit anti-Fgf21 (ab171941, Abcam), rabbit anti-MyHC I (22280-1-AP, Proteintech), rabbit anti-MyHC IIa (ab124937, Abcam), rabbit anti-MyHC IIb (20140-1-AP, Proteintech), and mouse anti-MyHC IIx (67299-1-Ig, Proteintech), rabbit anti-TGF-β1 (bs-0086R, Bioss, Beijing, China), rabbit anti-Smad2/3 (PA5-99539, Invitrogen), rabbit anti-p-p38 MAPK (8690, CST, MA, USA), mouse anti-Tubulin (T6199, Sigma-Aldrich, MO, USA), and mouse anti-β-actin (4967, CST). HRP-conjugated goat anti-mouse (CW0102S, CWBIO, Taizhou, China) or anti-rabbit (CW0156S, CWBIO) secondary antibodies were incubated at room temperature for 1 h. ECL (PEOO10, Solarbio) was used for enhanced chemiluminescence detection, according to the manufacturer’s instructions.
4
Western Blot Analysis of TGF-β1 Signaling
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Porcine GCs were lysed using RIPA lysis buffer (#BD0031, Bioworld) with 1% PMSF and protease inhibitor, and the total protein was extracted and collected for western blotting. The concentration of each protein sample was detected using the BCA method according to the manufacturer’s instructions. In brief, equal amounts of protein (~20 μg) from different samples were uploaded and separated by 4–20% SDS-PAGE. After electrophoresis for 1 h at 140 V, the separated proteins were transferred to PVDF membranes. The PVDF membranes were then incubated with blocking buffer containing 5% BSA and probed with the primary antibodies overnight. The next day, the membranes were resined with TBST buffer three times to remove excess primary antibodies, and then incubated with HRP-conjugated secondary antibodies. The high-solution images were obtained using a chemiluminescence imaging system, and the grayscale of each protein blot was quantified using ImageJ software. GADPH protein expression served as an internal control. The primary antibodies used were TGF-β1 (#bs-0086R, Bioss, 1:1000 dilution), SMAD3 (#D155234, Sangon, 1:1000 dilution), p-SMAD3 (#D155153, Sangon, 1:1000 dilution), and GAPDH (#D198662, Sangon, 1:2000 dilution).
5
Western Blotting Analysis of HIF-1α, CTGF, and TGF-β1
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6
Neonatal Skin Protein Expression
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At three time points (day 4, 7 and 14), the neonatal skin tissues of rats were collected for the immunohistochemical staining test. The total proteins were extracted from the neonatal skin tissues using the total protein extraction kit (Solarbio company, China) according to manufacturer’s instructions. The total protein solution of each group was added 50 µL 5 × loading buffer to boil for 10 min. Then, the protein samples from skin tissues of day 4, 7 and 14 were detected via western blotting. Each group of PVDF membranes were incubated by rabbit anti-bFGF, anti-TGF-β1, anti-VEGF and anti-β-actin polyclonal antibody antiserum (abcom, ab10420, Lot: GR1798 08, USA; Bioss, bs-0086R, Lot: AG07188387; Bioss, bs-4572R, Lot:AG08043197; Bioss, bs-0061R, Lot: AG07197903 China) and then the secondary antibody was incubated using goat anti-rabbit IgG antibody with alkaline phosphatase (Promega, USA). Finally, all images were visualized with AP developer (Promega, USA) for 1 min. This experiment was repeated three times.
7
Collagen and HDAC2 Regulation in Angiogenesis
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Erythromycin enteric-coated tablets (H42021990, Yichang Humanwell Pharmaceutical Co., LTD.); Vorinostat Capsules (180509, Beijing Hengrui Kangda Medical Science and Technology Development Co., Ltd.); Budesonide (AstraZeneca 8339000); Rabbit Anti-Collagen III Polyclonal Antibody (bs-10423R, Bioss); Rabbit Anti-Collagen I Polyclonal Antibody (bs-0549R, Bioss); Rabbit Anti-HDAC2 Polyclonal Antibody (bs-1813R, Bioss); Rabbit VEGF ELISA kit (MM-021001); Rabbit TGF-β1 ELISA kit (MM-3684001); Rabbit Polyclonal Anti-VEGF (bs-1313R, Bioss, 1/500–1/2000); Rabbit Polyclonal Anti-TGFβ1 (bs-0086R, Bioss, 1/500–1/2000); Rabbit monoclonal Anti-IL-8 (ab34100, abcam, 1/1000); Rabbit Polyclonal Anti-HDAC2 (OmnimAbs, OM105905, 1/500–1/2000); fluorescence microscope (CKX53, OLYMPUS); Microplate Reader (RT-6100, Rayto); Protein vertical electrophoresis instrument (DYY-6C, Beijing 61 instrument factory); Ultra High Sensitivity Chemiluminescence Imaging System (Chemi DocTM XRS+, Bio-Rad Shanhhai Laboratories).
8
Immunohistochemistry and Immunofluorescence Assay
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Immunohistochemical and immunofluorescence staining were performed by applying the method previously described [18 (link)]. Primary antibodies of TGF-β1 (bs-0086R, 1:400, Bioss) and CTGF (bs-0743R, 1:400, Bioss) were used for immunohistochemical staining. HIF-1α primary antibody (ab179483, 1:200, Abcam) was used for immunofluorescence staining.
9
Immunohistochemical Analysis of TGF-β and CCN2
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Tissues were fixed in neutral formaldehyde, embedded in paraffin and cut into 5-μm sections. Sections were subjected to routine immunohistochemical (IHC) staining as previously described[21 (link)]. Briefly, the sections were deparaffinized and rehydrated in a graded alcohol series, and antigen retrieval was performed in citrate buffer (pH 6.0) at 100°C for 15 min. After blocking with peroxide, the sections were incubated sequentially with a rabbit anti-human TGF-β polyclonal antibody (1:100, bs-0086R, Bioss, Beijing, China) and a goat anti-human CCN2 polyclonal antibody (1:100, sc-14939, Santa Cruz Biotechnology, Inc., USA) overnight at 4°C. Sections were incubated with the corresponding secondary antibodies conjugated to horseradish peroxidase at room temperature for 30 min. Finally, the sections were stained with diaminobenzidine (DAB), counterstained with hematoxylin, dehydrated, and cleared in xylene. As a negative control, the primary antibody was replaced with serum from non-immunized rabbit or goat.
10
Immunohistochemical Analysis of TGF-β1
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Tissue samples were embedded in paraffin and sectioned into slices with a thickness of approximately 5 μm. After dewaxing with xylene and dehydration in alcohol, antigen repair and blocking of endogenous peroxidase activity were performed. The sections were then incubated in primary antibodies against anti-TGF-β1 (bs-0086R, Bioss, China, 1:100) at 4 °C overnight. After rinsing three times with PBS, the tissue sections were incubated with secondary antibodies corresponding to the species for 1 h at room temperature. Then, 3,3′-diaminobenzidine tetrahydrochloride (DAB) color development solution was added. After staining with hematoxylin, the sections were visualized with a microscope. Positively stained areas were measured using ImageJ software (National Institutes of Health, Bethesda, USA).
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