Sections were baked, dewaxed and hydrated as previously described. The antigen was then repaired by proteinase K (0.3 mg/ml; Merck KGaA) treatment at 37°C for 30 min. Endogenous enzyme activity was extinguished using 3% hydrogen peroxide for 10 min. Slices were blocked with goat serum (cat. no. 5425; Cell Signaling Technology, Inc.) at 37°C for 1 h and incubated with primary antibody overnight at 4°C [rabbit anti-TGFβ1, cat. no. 3709, 1:100; rabbit anti-phosphorylated (p)-Smad2/3, cat. no. #8828S, 1:200; Cell Signaling Technology, Inc.; rabbit anti-a disintegrin and metalloproteinase with thrombospondin motifs 4 (Adamts4), cat. no. #ab185722, 1:200; Abcam], incubated with secondary antibody for 1 h at room temperature [horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody, cat. no. #7074, 1:1,000; Cell signaling Technology, Inc.; goat anti-rabbit lgG/Cy3, cat. no. bs-0295G-Cy3; 1:1,000; Bio-synthesis Inc.]. 3′-Diaminobenzidine (DAB, Bio-synthesis Inc.) was added for 5 min and then 4′, 6-diamidino-2-phenylindole or hematoxylin was used to counterstain the nuclei at room temperature for 5 min. The slides were observed under a light or fluorescence microscope (magnification, ×40, ×100 or ×200).
Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody is a detection reagent used in various immunoassay techniques. It consists of a goat-derived secondary antibody that is chemically linked to the enzyme horseradish peroxidase. This enzyme can catalyze a colorimetric reaction, enabling the visualization and detection of target proteins or antigens recognized by primary rabbit antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by Abcam (2 mentions)
Rabbit anti occludin, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Hrp color development kit, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Merck Group (1 mentions)
Rabbit anti tgf β1, by Cell Signaling Technology (1 mentions)
Luminata western hrp substrate, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Cyp2e1, by Abcam (1 mentions)
Carbon tetrachloride, by Merck Group (1 mentions)
Ketaved, by Vedco (1 mentions)
Acepromazine, by Vedco (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti α sma, by Abcam (1 mentions)
19 protocols using horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody
1
Immunohistochemical Analysis of TGF-β1 Signaling
2
Protein Expression Profile Analysis
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Total protein was extracted using RIPA buffer (Beyotime Biotechnology, China). Samples were separated by 14% SDS-PAGE, and then transferred onto PVDF membranes (Roche, Germany). After blocking in TBST (Tris buffered saline with 0.1% Tween-20) and 5% non-fat milk and incubated with primary antibodies for BDNF, ALP, OCN, COL1, Runx2, or β-actin (Cell Signaling Technology Inc., USA). Then the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Abcam, MA, USA). Immunoreactivity was detected by enhanced chemiluminescence reaction using LuminataTM Western HRP substrate (Millipore, USA). ECL images were acquired and analysed with the Chemiluminescence Imaging System (CLINX, Shanghai, China).
3
Molecular Mechanisms of Liver Injury
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Primary antibodies used include the following: CYP2E1 (Abcam, Cambridge, MA), MMP13 (clone LIPCO-IID1, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Clone 14C10, Beverly, MA). A horseradish peroxidase (HRP)-conjugated goat-anti-rabbit secondary antibody (Abcam) was used for the primary antibodies listed above. Olive oil and carbon tetrachloride were purchased from Sigma-Aldrich (St. Louis, MO), Buprenex analgesic (buprenorphine HCl) was manufactured by Reckitt Benckiser Healthcare (UK, Ltd, Hull England) and distributed by Reckitt Benckiser Pharmaceuticals, Inc. (Richmond, VA). Anesthetics used were from the following sources: ketamine (Akorn, Inc, Decator, IL), xylazine (KetaVed, VedCO, Inc., St. Joseph, MO), and acepromazine (VedCO, Inc.). Pyrimidine-4,6-dicarboxylic acid, bis-(4-fluoro-3-methyl-benzylamide) was used as a selective MMP13 inhibitor (EMD Millipore/Calbiochem, Billerica, MA, catalog number #444283).
4
Immunohistochemical Analysis of Kidney Tissue
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Following deparaffinization and rehydration, paraffin-embedded kidney sections were blocked with 3% hydrogen peroxide and subsequently incubated for 1 h at 37°C with the following rabbit primary antibodies: Anti-E-cadherin (1:25; catalog no. ab15148; Abcam, Cambridge, UK), anti-N-cadherin (1:100; catalog no. 13116; Cell Signaling Technology, Inc. Danvers, MA, USA), anti-α-SMA (1:200; catalog no. ab32575; Abcam, Cambridge, UK) and anti-vimentin (1:100; catalog no. 5741; Cell Signaling Technology, Inc.). They were subsequently incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:200; catalog no. ab6721; Abcam) for 1 h at room temperature. The reaction was visualized with a 3,3′-diaminobenzidine chromogen solution (Gene Tech Co., Ltd., Shanghai, China) and counterstained with hematoxylin. The slides were analyzed using Image-Pro Plus software version 6.0.
5
Western Blot Analysis of ABAT in HCC
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Eight pairs of HCC tissue samples were dissolved using radioimmunoprecipitation assay buffer (BIOSS, Beijing, China). A bicinchoninic acid protein assay kit (BIOSS, Beijing, China) was used to determine protein concentrations. Equivalent protein samples were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and diverted onto a polyvinylidene fluoride membrane (Millipore, USA). The membrane was incubated with primary rabbit anti-human ABAT antibody (1:2000 dilution; Abcam, USA) overnight at 4°C after blocking, followed by Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:4000 dilution; Abcam, USA) for 2 h. Glyceraldehyde-3-phosphate dehydrogenase was used as the control. In the end, the membrane was rinsed with phosphate-buffered saline with 0.1% Tween 20 buffer thrice and scanned with a gel imaging system (Thermo, Waltham, USA).
6
Nadroparin Protects Intestinal Barrier
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A total of 35 healthy 12-week-old female Sprague-Dawley (SD) rats (250–300 g) and 15 adult male SD rats (300–350 g) were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were maintained in a controlled environment (25±1°C, 50% humidity and 12-h light/dark cycle) and were given free access to water and a standard laboratory diet. The Medical Ethics Committee of Southeast University approved all animal studies.
The primary antibodies used in this study were: Rabbit anti-zonular occludens-1 (ZO-1) (cat. no. 61-7300; Invitrogen; Thermo Fisher Scientific, Inc.), rabbit anti-occludin (OCLN; cat. no. ab216327; Abcam), rabbit anti-NF-κB p65 (cat. no. ab16502; Abcam), rabbit anti-GAPDH (cat. no. ab9485; Abcam) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. ab130805; Abcam). The LMWH used was nadroparin calcium injection, which was purchased from GlaxoSmithKline plc. Bull serum albumin (BSA) was purchased from Gibco (Thermo Fisher Scientific, Inc.).
The primary antibodies used in this study were: Rabbit anti-zonular occludens-1 (ZO-1) (cat. no. 61-7300; Invitrogen; Thermo Fisher Scientific, Inc.), rabbit anti-occludin (OCLN; cat. no. ab216327; Abcam), rabbit anti-NF-κB p65 (cat. no. ab16502; Abcam), rabbit anti-GAPDH (cat. no. ab9485; Abcam) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. ab130805; Abcam). The LMWH used was nadroparin calcium injection, which was purchased from GlaxoSmithKline plc. Bull serum albumin (BSA) was purchased from Gibco (Thermo Fisher Scientific, Inc.).
7
Immunofluorescence Analysis of Tight Junction Proteins
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Immuno uorescence was analyzed by immunelabeling for ZO-1 and Occuludin. BeWo cells were seeded in 24-well plates and cultured in RPMI 1640 medium containing 10% FBS. After treated with different reagents at indicated concentrations for 24 h, the cells were incubated with rabbit anti-zonular occludens-1 (ZO-1) (1:1000, Invitrogen, Thermo Fisher Scienti c, Inc., USA) and rabbit anti-Occludin (1:2000, Abcam, Cambridge, UK) at 4˚C overnight, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:500, Abcam). The nucleus of each cell was stained with Hoechst 33342 (Blue). Images were taken of random elds at 200 × magni cation.
8
Histological Assessment of Liver Injury
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Liver tissues were fixed in 10% formalin and dehydrated. Paraffin-embedded sections (4 μm) were stained with H&E or Masson’s trichrome. For immunohistochemical staining, the sections were incubated with a primary antibody against 4-HNE (1:100), F4/80 (1:100), CD4 (1:500), collagen I (1:100), or fibronectin (1:100). These antibodies were purchased from Abcam (Cambridge, UK), except for F4/80 (Santa Cruz Biotechnology Inc., Dallas, TX, USA). After washing, the sections were probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000; Abcam, Cambridge, UK). Images were captured using a confocal microscope (Nikon, Tokyo, Japan). The percentage of positively stained area was analyzed in 5 randomly selected fields per liver sample, using i-Solution DT software (IMT i-Solution Inc., Coquitlam, BC, Canada). The number of F4/80 or CD4-stained cells was counted in 5 randomly selected fields per liver sample.
9
Immunofluorescence Analysis of Tight Junction Proteins
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Immuno uorescence was analyzed by immunelabeling for ZO-1 and Occuludin. BeWo cells were seeded in 24-well plates and cultured in RPMI 1640 medium containing 10% FBS. After treated with different reagents at indicated concentrations for 24 h, the cells were incubated with rabbit anti-zonular occludens-1 (ZO-1) (1:1000, Invitrogen, Thermo Fisher Scienti c, Inc., USA) and rabbit anti-Occludin (1:2000, Abcam, Cambridge, UK) at 4˚C overnight, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:500, Abcam). The nucleus of each cell was stained with Hoechst 33342 (Blue). Images were taken of random elds at 200 × magni cation.
10
Western Blot Analysis of Protein Expression
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The protein expression in whole cell lysates or nuclear extracts was analyzed using western blot analysis. The radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) was used to extract total protein from B3 cells. Next, a BCA protein assay kit (Beyotime) was used to quantify the proteins. Then, 30-µg protein samples were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were then blocked using 5% skimmed milk for 1 h at room temperature, followed by incubation with primary antibodies against Bcl-2 (Abcam, Cambridge, USA), Bax (Abcam), Nrf2 (Cell Signaling Technology, Beverly, USA), HO-1 (Abcam), NQO1 (Cell Signaling Technology), and GAPDH (Cell Signaling Technology) at 4°C overnight. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Abcam) for 1 h at room temperature, followed by exposure to the enhanced chemiluminescent reagent (Pierce, Rockford, USA). The intensity of proteins signals was quantified using Quantity One software v. 4.1.1 (Bio-Rad Laboratories, Hercules, USA).
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