A BCA kit (Beyotime, Shanghai, China) was utilized in order to quantify the amount of protein. Equal amount of protein preparations was run on SDS-polyacrylamide gels, electrotransferred to polyvinylidene difluoride membranes, and blotted with anti-P65 (Cell Signaling Technology, Boston, USA) overnight at 4°C using slow rocking. Then, they were blotted with HRP-conjugated secondary antibody (Servicebio, China). The antibody-antigen complexes were visualized by using the electrochemiluminescence (ECL) kit (Servicebio, China). The protein bands were detected with ChemiScope 6300 (Clinx Science Instruments Co. Ltd, China). The bands were analyzed with the AlphaEaseFC software and compared with β-actin.
Hrp conjugated secondary antibody
Manufactured by Wuhan Servicebio Technology
Sourced in China
HRP-conjugated secondary antibodies are a type of laboratory reagent used in various immunoassay techniques. They are designed to bind to primary antibodies, with the horseradish peroxidase (HRP) enzyme attached to the secondary antibody. The HRP enzyme can then be used to catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Gb13030 2, by Wuhan Servicebio Technology (2 mentions)
Ecl kit, by Wuhan Servicebio Technology (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Bca kit, by Beyotime (1 mentions)
Chemiscope 6300, by Clinx (1 mentions)
Ki67 cell proliferation kit, by Sangon (1 mentions)
Ab154532, by Proteintech (1 mentions)
Ab195352, by Fortis Life Sciences (1 mentions)
Chemiluminescent reagent, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab0037, by Abcam (1 mentions)
Gb23302, by Wuhan Servicebio Technology (1 mentions)
Total protein extraction kit, by BestBio (1 mentions)
Phosphatase inhibitor cocktail, by CWBIO (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Light microscopy, by Olympus (1 mentions)
Fluorescent microscope system, by Olympus (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
23 protocols using hrp conjugated secondary antibody
1
Western Blot Protein Quantification
2
Evaluating Tumor Proliferation and Angiogenesis
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Tumor sections (4 μm) were prepared and stained with PCNA and CD31 antibodies for the purpose of evaluating tumor proliferation and tumor angiogenesis in different treatment groups. Briefly, the paraffin-embedded tumor tissue sections underwent de-paraffinage and rehydration. The antigen retrieval process was conducted, followed by endogenous peroxidase inactivation with a 3% hydrogen peroxide solution. Then, the tumor sections were punched with Triton X-100 (for PCNA staining), blocked with goat serum, and sequentially incubated with PCNA antibody or CD31 antibody (Servicebio, China) and corresponding HRP conjugated secondary antibody (Servicebio, China). Finally, the labeled sections were colorated by DAB and counterstained with hematoxylin. All stained sections were observed under a light microscope at 200× magnification. Cells with positive expression of PCNA or CD31 were stained in yellow brown. The PCNA index was calculated as the amount of PCNA staining positive cells divided by the amount of total cells in five randomly selected fields. The microvascular density was calculated as the amount of CD31 staining positive cells in five randomly selected fields.
3
Ki67 Immunostaining Protocol for Cell and Tumor Samples
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For cultured cells, Ki67 staining was carried out using a Ki67 Cell Proliferation Kit (Sangon Biotech, China) according to the manufacturer's recommendations. In brief, cells were rinsed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 10 min. After blocking with 10% FBS in PBS for 1 h, cells were incubated with a rabbit anti-Ki67 antibody (1:100, Sangon Biotech) overnight at 4°C followed by an incubation with a Cy3-conjugated goat anti-rabbit IgG (red, 1:100, Sangon Biotech) for 1 h. Nuclear were counterstained with DAPI (blue).
For tumors from mice model, Ki67 were detected by standard immunohistochemistry protocols. Slides were deparaffinized, hydrated and boiled in EDTA buffer (pH 9.0) for 2 min for antigen retrieval. After treated with 3% H2O2 for 25 min, slides were blocked with 3% BSA for 30 min, and then incubated with a rabbit anti-Ki67 antibody (1:100, GB13030-2, Servicebio, China) overnight at 4°C, followed by an incubation with HRP-conjugated secondary antibody (Servicebio) for 50 min. Then, the signal was developed by in DAB (brown) solution for 5 min and the nuclear were counterstained with hematoxylin (blue).
For tumors from mice model, Ki67 were detected by standard immunohistochemistry protocols. Slides were deparaffinized, hydrated and boiled in EDTA buffer (pH 9.0) for 2 min for antigen retrieval. After treated with 3% H2O2 for 25 min, slides were blocked with 3% BSA for 30 min, and then incubated with a rabbit anti-Ki67 antibody (1:100, GB13030-2, Servicebio, China) overnight at 4°C, followed by an incubation with HRP-conjugated secondary antibody (Servicebio) for 50 min. Then, the signal was developed by in DAB (brown) solution for 5 min and the nuclear were counterstained with hematoxylin (blue).
4
Protein Extraction and Western Blot Analysis
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Protein solubilization buffer was used for cell lysis. Protein extracts were performed according to the manufacturer’s instructions of Total Protein Extraction Kit (BestBio, Shanghai, China). SDS‒PAGE was using for separating the proteins, after that, proteins were transferred to nitrocellulose membranes (Millipore). Antibodies used in our study were mainly anti-GAPDH (Abways, AB0037), anti-NCBP1(Abcam ab154532, Proteintech 10349-1-AP), anti-METTL3 (Abcam ab195352, BETHYL A301-568A) and anti-MYC (Proteintech 10828-1-AP, Cell Signaling Technology #9402). GAPDH was served as a loading control. Finally, membranes were probed by HRP-conjugated secondary antibody (Servicebio GB23302) and visualized by using chemiluminescent reagents (Millipore, Merck, Darmstadt, Germany).
5
Western Blot Analysis of GAP43 Expression
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Adherent cells were collected by gentle scraping, washed three times in phosphate-buffered saline (PBS), and lysed in Nonidet P 40 cell lysis buffer supplemented with phosphatase inhibitor cocktail (Cwbio, Beijing, China) and protease inhibitor cocktail (Roche, Mannheim, Germany). A Bradford calorimetric assay (Cwbio) was used to measure the protein concentration of the cell lysates. Thirty micrograms of total protein was separated on a 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Each membrane was blocked in TBS-Tween-20 (TBS-T) containing 5% non-fat milk at room temperature for 2 h, followed by incubation with anti-GAP43 antibody (1:1000, Huabio, Hangzhou, China) at 4 °C overnight. The next day, the membrane was incubated with an HRP-conjugated secondary antibody (Servicebio, Wuhan, China) at room temperature for 2 h, and the results were detected using an enhanced chemiluminescence (ECL) reagent (Cwbio). Tubulin (1:1000; Antibody Revolution, San Diego, CA, USA) was used as the loading control.
6
Immunohistochemical Analysis of Intervertebral Disc
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The methods have been reported previously [13 (link)]. Briefly, intervertebral disc sections were firstly embedded in paraffin. Then, the sections were deparaffinized, and the 3% hydrogen peroxide was used to block the endogenous peroxidase. Subsequently, after antigen retrieval using pepsin (Servicebio, Wuhan, China) in 5 mM HCl, the sections were incubated with 10% goat blocking serum for 20-30 min, then with primary antibody against IL-1β (#12242, 1 : 100, Cell Signaling Technology, Inc. USA), p65 (#8242, 1 : 400, Cell Signaling Technology, Inc. USA), and Nrf2 (340675, Zenbio, Chengdu, China, 1 : 500) at 4°C overnight. Finally, HRP-conjugated secondary antibody (GB23302/23303, Servicebio, Wuhan, China) was added to the sections, and the sections were further counterstained using hematoxylin. Images were obtained using light microscopy (Olympus, Japan).
7
Immunohistochemical Analysis of Vulvar Caspase-1
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To assess the pathological changes, vulvar paraffin sections were stained with haematoxylin and eosin. Immunohistochemistry was used to detect the expression of caspase-1. Tissue sections were dewaxed, hydrated, antigen repaired, membrane permeated, and blocked. After incubating with primary antibodies against caspase-1 and HRP-conjugated secondary antibody (Servicebio, Wuhan, China), the tissue sections were visualized by DAB, and counterstaining was performed with haematoxylin. For immunofluorescence staining, the slides followed the same procedure as immunohistochemistry until antigen was repaired. After blocking with 10% normal goat serum for 1 h, the tissue sections were incubated with primary antibodies and Alexa Flour 488 or Cy3-conjugated secondary antibody. The nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI). The images were detected by Olympus fluorescent microscope system.
8
Adipose Tissue Immunohistochemistry
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Briefly, adipose mass was fixed in 4% PFA, dehydrated in 30% sucrose solution, and embedded in OCT. Samples were then sectioned (10-μm-thick sections) and incubated with GFP antibody (1:100, Servicebio, Wuhan, China) overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibody (1:200, Servicebio, Wuhan, China) at room temperature for 1 h away from light. Cells were then visualized and imaged using a microscope.
9
Histological Analysis of Liver Tissue
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Sections of liver tissues were fixed in 10% formalin for paraffin embedding. Paraffin-embedded liver sections were subjected to hematoxylin and eosin staining.
For immunohistochemical analysis, liver tissue sections were permeabilized with blocking buffer (3% bovine serum albumin) for 1 h after antigen retrieval and 3% H2O2 incubation. Then, the liver sections were incubated in Ly6G antibodies (1:1000 dilution; Servicebio, #GB11229) overnight at 4 °C. After washing with PBS, samples were incubated with HRP-conjugated secondary antibody (1:1000 dilution; Servicebio, #GB23303) 1 h at 37 °C prior to the incubation with Diaminobenzidine Tetrahydrochloride (DAB). Section images of H&E and IHC staining were visualized by a light microscope. Cell death was analyzed using the Dead End Fluorometric Tunel System (Promega, #G3250) according to the manufacturer's instructions. Signals of dead cells were visualized by fluorescence microscopy. Necrotic area, Ly6G+ neutrophils, and Tunel+ cells were quantified using Image J software.
For immunohistochemical analysis, liver tissue sections were permeabilized with blocking buffer (3% bovine serum albumin) for 1 h after antigen retrieval and 3% H2O2 incubation. Then, the liver sections were incubated in Ly6G antibodies (1:1000 dilution; Servicebio, #GB11229) overnight at 4 °C. After washing with PBS, samples were incubated with HRP-conjugated secondary antibody (1:1000 dilution; Servicebio, #GB23303) 1 h at 37 °C prior to the incubation with Diaminobenzidine Tetrahydrochloride (DAB). Section images of H&E and IHC staining were visualized by a light microscope. Cell death was analyzed using the Dead End Fluorometric Tunel System (Promega, #G3250) according to the manufacturer's instructions. Signals of dead cells were visualized by fluorescence microscopy. Necrotic area, Ly6G+ neutrophils, and Tunel+ cells were quantified using Image J software.
10
Immunohistochemical Analysis of Tumor Markers
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For tumors from mice model, Ki67, p-Akt, and Cleaved Caspase3 were detected by standard immunohistochemistry protocols. Slides were deparaffinized, hydrated, and boiled in EDTA buffer (pH 9.0) for 2 min for antigen retrieval. After treated with 3% H2O2 for 25 min, slides were blocked with 3% BSA for 30 min, and then incubated with a rabbit antibody (1:100, GB13030-2, Servicebio, China) overnight at 4 °C, followed by an incubation with HRP-conjugated secondary antibody (Servicebio) for 50 min. Then, the signal was developed by in DAB (brown) solution for 5 min and the nuclear was counterstained with hematoxylin (blue).
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