Before immunostaining, tissue sections were deparaffinized and rehydrated, and antigen retrieval was performed in citrate buffer (pH 6). The sections were then treated with 0.3% hydrogen peroxide to block endogenous peroxidase activity. After being washed in PBS, tissues were blocked with 5% normal goat serum for 1 h and stained with the following primary antibody overnight: Muc2 (1:1,000, Proteintech), Ki67 (1:50, Abcam, Cambridge, MA, USA), Lgr5 (1:400, Affinity Biosciences, Cincinnati, OH, USA), Caspase3 (1:2,000, Cell Signaling Technology, Danvers, MA, USA), Occluding (1:200, Abcam), CCL7 (1:200, Bioss), p-IκBα (1:200, Affinity Biosciences), HIF1α (1:100, Affinity Biosciences), or CYP1A1 (1:200, Proteintech). The sections were repeatedly washed with PBS and stained with horseradish peroxidase (HRP)-labeled secondary antibody (Zsbio, Beijing, China) for 30 min at room temperature. Bound antibodies were measured with diaminobenzidine chromogenic substrate (Zsbio). The sections were finally counterstained with hematoxylin, dehydrated, and mounted. Tissues were examined and imaged using an Olympus BX46 microscope. The intensity of the staining was analyzed by the software Image Pro Plus.
Horseradish peroxidase hrp labeled secondary antibody
Manufactured by ZSGB-BIO
Sourced in China
Horseradish peroxidase (HRP)-labeled secondary antibody is a conjugated antibody used in various immunoassay techniques. It consists of a secondary antibody that binds to the primary antibody, with the horseradish peroxidase enzyme attached. The HRP enzyme can catalyze a colorimetric reaction, enabling the detection and visualization of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Cell Signaling Technology (3 mentions)
Diaminobenzidine chromogenic substrate, by ZSGB-BIO (2 mentions)
Bx46 microscope, by Olympus (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Protease inhibitor cocktail tablet, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Loading buffer, by Beyotime (2 mentions)
Occluding, by Abcam (1 mentions)
Cyp1a1, by Proteintech (1 mentions)
Hif 1α, by Affinity Biosciences (1 mentions)
P iκbα, by Affinity Biosciences (1 mentions)
Pho irf3, by Abcam (1 mentions)
Beta actin, by ZSGB-BIO (1 mentions)
Gpr43, by Abcam (1 mentions)
Gpr41, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Occludin, by Abcam (1 mentions)
Il 13rα2, by Proteintech (1 mentions)
Nf κb, by Proteintech (1 mentions)
7 protocols using horseradish peroxidase hrp labeled secondary antibody
1
Immunohistochemical Analysis of Intestinal Markers
2
Western Blot Analysis of Apoptosis Markers
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Cells were lysed in RIPA buffer (Beyotime, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). The protein concentration was quantified using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of protein were denatured with loading buffer (Beyotime, China) at 100°C for 10 min, subjected to 12% SDS-PAGE, and then blotted to a methanol-activated PVDF membrane (Millipore, USA). After blocked with 5% bovine serum albumin (ABCONE, China) in tris-buffered saline (TBS) for 1 h at room temperature, the membranes were respectively incubated overnight with the following antibodies at 4°C: MAVS (1 : 1000, Abcam, USA); pho-IRF3 (1 : 1000, Abcam, USA); caspase-3 (1 : 1000, Cell Signaling Technology, USA); IFN-I, IRF3, cyto C, Bax, and Bcl2 (1 : 1000, Affinity Biosciences, USA); and beta-actin (1 : 1000, ZSGB-BIO, China). After being washed for three times with TBS, the membranes were incubated with the horseradish peroxidase- (HRP-) labeled secondary antibody (1 : 2500, ZSGB-BIO, China) for 1 h at room temperature.
3
Immunohistochemical analysis of intestinal markers
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For immunohistochemical staining and imaging, tissue sections were deparaffinized, dehydrated, and subjected to antigen retrieval in citrate buffer (pH 6). The sections were then exposed to 0.3% hydrogen peroxide to block endogenous peroxidase activity, blocked with 5% normal goat serum for 1 h, and stained overnight with primary antibodies against Muc2 (1:1000, Proteintech, Chicago, IL, USA), Ki67 (1:50, Abcam, Cambridge, MA, USA), Lgr5 (1:400, Affinity Biosciences, Cincinnati, OH, USA ), Caspase3 (1:2000, Cell Signaling Technology, Danvers, MA, USA), ZO-1 (1:500, Thermo Fisher Scientific, Waltham, MA, USA), Occludin (1:200, Abcam), GPR41 (1:1000, Abcam), GPR43 (1:500, Abcam), β-catenin (1:200, Cell Signaling Technology), or p-p38 (1:200, Cell Signaling Technology). The sections were washed at least five times with PBS and stained for 30 min with horseradish peroxidase (HRP)-labeled secondary antibody (Zsbio, Beijing, China). The staining was developed with diaminobenzidine chromogenic substrate (Zsbio). The sections were finally counterstained with hematoxylin, dehydrated, and mounted. Tissues were imaged using an Olympus BX46 microscope. Staining intensity was analyzed using the software Image Pro Plus.
4
Western Blot Analysis of Radiation-Induced Signaling
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Cells were pretreated with or without 20 μM ISO for 24 h followed by 2 Gy X-rays irradiation. After 12 h, cells were lysed with RIPA buffer (Beyotime, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). Equal amounts of total protein were denatured with 1× loading buffer (Beyotime, China) and subjected to 12% SDS-PAGE and then transferred to a methanol-activated PVDF membrane (Millipore, United States). The membranes were blocked and then probed overnight at 4°C with the following primary antibodies against: Bax, Bcl2, NF-κB, IL-13Rα2, and GAPDH (1:1,000, Proteintech, China); IL-13, IL-13Rα1, (1:1,000, Affinity Biosciences, United States); p-IκBα and p-NF-κB (1:800, CST, United States). After three washes with TBS, membranes were probed with the horseradish peroxidase- (HRP-) labeled secondary antibody (1:2,500, ZSGB-BIO, China) for 1 h at room temperature. Staining was visualized using enhanced chemiluminescence (ECL) reagents, according to the manufacturer’s recommendations. The intensity of protein bands on the western blot image was quantified by Image J software.
5
Western Blot Analysis of Protein Samples
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The total cell lysates were harvested using a 2% SDS lysis buffer on ice. After the determination of protein concentration, the protein samples were loaded into wells for electrophoresis through 8% or 12% SDS-PAGE gels. The protein bands were separated and transferred into a nitrocellulose membrane (Millipore, Ireland). Then, the membrane was blocked in 5% fat-free milk for 60 min and incubated overnight at 4°C with the primary antibodies (Table S2 ). The next day, the membranes were washed three times with Tris-buffered saline and Tween 20 (TBST) and then incubated for 60 min in horseradish peroxidase (HRP)-labeled secondary antibody (1:5,000, Zsbio, Beijing, China). The bands were visualized using an enhanced chemiluminescence (ECL) Kit (Applygen, China) in a Tanon 5200 Multi-imaging system (Tanon, China).
6
Total Protein Extraction and Western Blot Analysis
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Total protein was extracted by Tissue or Cell Total Protein Extraction Kit (KeyGen Biotech, China) according to the manufacturer’s instructions. BCA Protein Assay Kit (Cwbio, China) was used to quantify the protein abundance. Samples with equal volume of protein were subjected to 10% SDS-PAGE (Beyotime, China) and then transferred to the polyvinylidene fluoride (PVDF) membranes (EMD Millipore, USA). The membranes were then blocked with Tris-buffered saline 0.1% Tween-20 (TBST) containing 6% non-fat milk at room temperature, and then incubated with rabbit anti-PHF2 (1:1,000, 24624-1-AP, Proteintech, China) and rabbit anti-GAPDH (1:1,000, 10494-1-AP, Proteintech, China) antibodies at 4°C overnight. Horseradish peroxidase (HRP)-labeled secondary antibodies (1:5,000, ZSGB-BIO, China) were used to obtain the signals from proteins. Finally, the protein was visualized with an electrochemiluminescence (ECL) system (Biorad, USA) using ECL reagents (Advansta, USA).
7
Western Blot Analysis of Autophagy and Apoptosis Markers
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According to the manufacture's instruction, RIPA lysis buffer (Pulitzer, Beijing, China) was applied to cells and protein concentrations calculated using the BCA method (ComWin Biotech Co.,Ltd, Beijing, China) [35 (link)]. Proteins were subjected to SDS-PAGE electrophoresis (Beyotime Biotechnology, China), transferred to PVDF membrane (Millipore Company, USA) at 250mA, and blocked in 3% BSA for 1h. The antibodies used were: primary LC3 antibody (Cell Signaling, USA, 4108), primary Notch1 antibody (Cell Signaling, USA, 3608), primary Beclin antibody (Cell Signaling, USA, 3738), primary P62 antibody (Abcam, UK, ab56416), primary p53 antibody (Santa Cruz Biotechnology, USA, sc-126), primary Bcl-2 antibody (Santa Cruz Biotechnology, USA, sc-7382), and anti β-actin antibody (Santa Cruz Biotechnology, USA, sc-130619). Antibodies were incubated at 4°C overnight. Membranes were washed with TBST (10 min ×3), horseradish peroxidase (HRP) labeled secondary antibodies (ZSGB-BIO, Beijing, China, ZB-2301) were added, and membranes were incubated at room temperature for 1h. A chemiluminescent substrate (Pulitzer, Beijing) was applied to develop films using an ImageQuant LAS 4000 mini Ultrasensitive chemiluminescence imager.
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