Goat anti rabbit igg h l hrp antibody

Manufactured by Abcam

Sourced in United Kingdom

The Goat Anti-Rabbit IgG H&L (HRP) antibody is a secondary antibody that binds to rabbit primary antibodies. The antibody is conjugated with horseradish peroxidase (HRP), which can be used for detection and visualization applications.

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7 protocols using goat anti rabbit igg h l hrp antibody

Lung Tissue Histological and IHC Analysis

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The lung tissue was fixed in 10% formalin, embedded and sectioned (4 μm) for hematoxylin and eosin (HE) and immunohistochemistry (IHC). Lung tissue sections were dewaxed with xylene, hydrated with ethanol, and sequentially stained with HE. Images were taken from five different fields using a fluorescence pathological microscope (Olympus */BX53 + DP80, Japan), and Image-Pro Plus software 6.0 (Media Cybernetics) was used to measure the mean linear intercept (MLI) for each group, as an estimate of alveolar diameter.
The expression levels of P53 and P21 were determined by IHC. Briefly, lung tissue sections were deparaffinized with xylene, hydrated with alcohol, repaired with EDTA and then incubated with antibody against mouse p53 (Abclone, Wuhan, China) and p21 (Abclone, Wuhan, China) overnight at 4°C. On the following day, the sections were incubated with secondary Goat Anti-Rabbit IgG H&L (HRP) antibody (Abcam, Cambridge, UK) and stained with diaminobenzidine (Solarbio, DA1010, China). Images of lung sections were captured and analyzed as described above.

Xiaofei Y., Tingting L., Xuan W, & Zhiyi H. (2022). Erythromycin attenuates oxidative stress-induced cellular senescence via the PI3K-mTOR signaling pathway in chronic obstructive pulmonary disease. Frontiers in Pharmacology, 13, 1043474.

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Tissue Microarray Analysis of CACYBP and Ki67 in PC

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Tissue microarray of PC patients was obtained from Shanghai Outdo Biotech Company (#JS W-11-01), which included 87 tumor tissues and 45 para-carcinoma tissues. Ethical approval was obtained from the Ethics Committee of First Affiliated Hospital, School of Medicine, Shihezi University. In brief, tumor specimens were dewaxed by xylene for 15 min and rehydrated by 100% alcohol for 10 min. The antigen was repaired in 0.01 M sodium citrate buffer (pH = 6.0) at 180 ℃ for 5 min and blocked by 3% hydrogen peroxide bath for 15 min. After washing, the sections were incubated with anti-CACYBP (1: 50, Cat. # ab171972, Abcam) or anti-Ki67 (1:200, Cat. # ab16667, Abcam) overnight at 4 ℃ and followed by conjugation to the Goat Anti-Rabbit IgG H&L (HRP) antibody (1: 400, Abcam, USA, Cat. # ab6721) at room temperature for 2 h. Tissue sections were stained with DAB and hematoxylin, and then exanimated with microscopic (Olympus). All specimens were classified into categories based on staining percentage and staining intensity as previously described (Feng et al. 2021 (link)).

Li Q., Liu Z., Ma L., Yin W, & Zhang K. (2022). CACYBP knockdown inhibits progression of prostate cancer via p53. Journal of Cancer Research and Clinical Oncology, 149(9), 5761-5772.

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Western Blot Analysis of FXR, DHRS9, and ATP Pathway

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Radioimmunoprecipitation buffer (Thermo Fisher Scientific, USA) was used to lyse the cells. Cell lysate containing 50 μg of total protein was transferred onto a polyvinylidene fluoride membrane (Millipore, USA) after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific, USA). The membrane and primary antibody were incubated at 4°C overnight. The protein bands were then rinsed with Tris buffer saline plus Tween (TBST) buffer 3 times, 10 min each. Next, the membrane and secondary antibody were incubated at room temperature for 2 h. Chemiluminescence substrate (Thermo Fisher Scientific, USA) was added to observe the protein bands.
The primary antibodies including Anti-FXR (ab129089, diluted at 1 : 1000), anti-DHRS9 (ab126074, diluted at 1 : 1000), anti-ATP5D (ab97491, diluted at 1 : 1000), anti-ATP5E (Cat #PA5-104424, diluted at 1 : 1000), anti-NDUFA3 (H00004696-K, diluted at 1 : 1000), and anti-GAPDH (ab9485, diluted at 1 : 2500) were all rabbit-derived antibodies. Anti-FXR, anti-DHRS9, anti-ATP5D, and anti-GAPDH were from Abcam (UK). Anti-ATP5E antibody was from Thermo Scientific (USA). The anti-NDUFA3 antibody was bought from Abnova (China). Goat anti-rabbit IgG H&L (HRP) antibody (Abcam, ab6721, diluted at 1 : 2000, UK) served as the secondary antibody.

Zhao J., Wang Y., Wang Y., Gao J., Yang H., Wu X, & Li H. (2022). Transcription Factor FXR Activates DHRS9 to Inhibit the Cell Oxidative Phosphorylation and Suppress Colon Cancer Progression. Analytical Cellular Pathology (Amsterdam), 2022, 8275574.

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Rab3A Protein Expression in Spermatozoa

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Rab3A protein expression in spermatozoa was determined using an enzyme-linked
immunosorbent assay. Spermatozoa were treated with rehydration buffer to extract
total proteins [7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl)
dimethylammonio]-1-propane sulfonate, 1% (w/v) octyl b-D-glucopyranoside,
24 mM phenylmethylsulfonyl fluoride, 1% (w/v) dithiothreitol,
0.05% (v/v) Triton X-100, and 0.002% (w/v) bromophenol blue] at
4°C for 1 h. Protein concentration was measured following the Bradford
protein-binding protocol (Bradford, 1976). Extracted protein (50 μg) was
loaded into 96-well plates and incubated overnight at 4°C. The plates
were then washed with PBS containing 0.05% Tween-20 (PBST) and blocked
with a blocking solution [1% (w/v) bovine serum albumin in PBST] for 90
min at 37°C. Subsequently, the plates were incubated with anti-Rab3A
antibody (1:5,000; Abcam, Cambridge, UK) for 90 min at 37°C, followed by
incubation with goat anti-rabbit IgG H&L (HRP) antibody (1:5,000; Abcam)
for 90 min at 37°C. Tetramethylbenzidine solution was used to activate
peroxidase for 15 min at room temperature. Activation was terminated with 1 N
sulfuric acid, and Rab3A protein signals were detected at 450 nm using a
microplate reader (Gemini Em; Molecular Devices, Sunnyvale, CA, USA).

Jang S.I., Jo J.H., Claudine U., Jung E.J., Lee W.J., Hwang J.M., Bae J.W., Kim D.H., Yi J.K., Ha J.J., Oh D.Y, & Kwon W.S. (2024). Correlation between Rab3A Expression and Sperm Kinematic Characteristics. Development & Reproduction, 28(1), 13-19.

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Immunohistochemical Analysis of TLR9, Myd88, and p-NF-κB

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IHC studies are used to detect the expressions of TLR9, myeloid differentiation primary response protein-88 (Myd88), phosphorylated p nuclear factor κB (p-NF-κB/ p-p65) as described previously. 33 (link) Briefly, the sections are washed three times with PBS and endogenous peroxidases are removed by incubation for 10 min at room temperature. Afterward, the sections are incubated with primary antibodies Myd88 (Cat. ab133739, Abcam), TLR9 (Cat. ab37154, Abcam), p-NF-κB (Cat. ab239882, Abcam) overnight at 37°C and then incubated with the secondary Goat Anti-Rabbit IgG H&L (HRP) antibody (Cat. ab6721, Abcam) for 2 h at room temperature. The slides are prepared with DAB and analyzed by Olympus BX43 (X 200).

Li S., Feng G., Zhang M., Zhang X., Lu J., Feng C, & Zhu F. (2022). Oxymatrine attenuates TNBS-induced colinutis in rats through TLR9/Myd88/NF-κB signal pathway. Human & experimental toxicology, 41.

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Western Blot Analysis of FPR1 Protein

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Homogenized tissue specimens and cells were lysed directly in a RIPA lysis buffer (Pierce Biotechnology, Rockford, IL, USA) with both a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland) and Halt phosphatase inhibitor cocktail (Pierce Biotechnology). A mixture containing NuPAGE LDS Sample Buffer, NuPAGE Sample Reducing Agent (Life Technologies, Carlsbad, CA, USA) and distilled water were added to lysates. The samples were heated to 70°C for 10 min, and equal amounts of protein were loaded into a NuPAGE Novex 4–12% Bis-Tris gel (Life Technologies). Gel electrophoresis and blotting onto a PVDF membrane (Life Technologies) were performed according to the NuPAGE Technical Guide (Invitrogen). Tris buffered saline with 0.1% Tween-20 (Sigma Aldrich) and 5% Bio-Rad Blotting-grade blocker (Bio-Rad, Hercules, CA, USA) were used for blocking, while primary and secondary antibodies were diluted in the blocking buffer. Membranes were probed with antibodies against FPR1 and β-actin (both from Abcam, Cambridge, UK) and concentrations of the antibodies were 1:500 and 1:5000 respectively. Goat Anti-Rabbit IgG H&L (HRP) antibodies (Abcam, Cambridge, UK) at a 1:5000 dilution were used as secondary antibodies. SuperSignal West Pico Chemiluminescent Substrate (Pierce-Thermo scientific, Rockford, IL) was used for detection, and images were acquired on a Fujifilm LAS-3000 Imager.

Øie C.I., Snapkov I., Elvevold K., Sveinbjørnsson B, & Smedsrød B. (2016). FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides. PLoS ONE, 11(8), e0160602.

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Protein extraction and Western blot analysis

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Cell proteins were extracted in RIPA buffer (Sigma-Aldrich; Merck KGaA). Nuclear proteins were extracted using CelLytic™ NuCLEAR™ reagent (Sigma-Aldrich; Merck KGaA). Proteins were quantified using a BCA assay, and equal concentrations (50 µg/lane) were resolved on 12.5% polyacrylamide gels at 130 V. Proteins were transferred to PVDF membranes and blocked in 2.5% skimmed milk at 37˚C for 1 h. Membranes were probed with primary antibodies overnight at 4˚C, including anti-rabbiti-TLR4 (1:1,000; cat no. 13867; Abcam), anti-rabbit-NF-κB p65 (1:1,000; cat no. 3034; Cell Signaling Technologies, Inc.), phospho-NF-κB p65 (1:1,000; cat no. 3033; Cell Signaling Technologies, Inc.), anti-rabbit-Lamin B (1:2,000; cat no. 194109; Abcam) and anti-rabbit-β-actin (1:2,000; cat no. 115777; Abcam). Membranes were washed in TBS containing 0.1% Tween 20 and labeled with Goat Anti-Rabbit IgG H&L (HRP) antibodies (1:3,000; cat no. 7090; Abcam) for 1 h at room temperature. Membranes were then washed and proteins were visualized using the ECL detection system (Amersham; Cytiva) on a ChemiDoc Bio-Rad system (Bio-Rad Laboratories, Inc.). Band intensities were semi-quantified using ImageJ software v.1.52v (National Institutes of Health).

Ye Y., Wang P, & Zhou F. (2021). miR-489-3p inhibits TLR4/NF-κB signaling to prevent inflammation in psoriasis. Experimental and Therapeutic Medicine, 22(1), 744.

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