Kidney tissues were fixed in 4% neutral buffered formalin, dehydrated in graded sucrose, and embedded in paraffin. Immunohistochemical staining of p53 was performed using a standard biotin-streptavidin-peroxidase method. Briefly, 4 μm thick sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked by 5% BSA for 30 min at room temperature, then the sections were incubated with the primary antibody for p53 (rabbit, 1:100; Protein Tech Group, Chicago, IL, USA) overnight at 4 °C. Then, sections were washed in PBS and incubated with biotinylated goat anti-rabbit antibody and HRP-labeled streptavidin (Beyotime, Jiangsu, China), each for 20 min. At last, peroxidase activity was visualized by diaminobenzidine, and sections were counterstained with hematoxylin and observed under light microscope and 20 glomeruli were randomly chosen for counting the p53-positive cells as described previously44 (link).
Hrp labeled streptavidin
Manufactured by Beyotime
Sourced in China, United States
HRP-labeled streptavidin is a protein complex composed of streptavidin conjugated with horseradish peroxidase (HRP). Streptavidin has a high affinity for biotin, and the HRP enzyme can catalyze colorimetric or chemiluminescent reactions. This product is commonly used in various biotechnological and immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti human iga1 antibody, by Santa Cruz Biotechnology (2 mentions)
96 well immunoplates, by Corning (2 mentions)
Hematoxylin, by Solarbio (2 mentions)
3 3 diaminobenzidine (dab), by Solarbio (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Biotin, by Merck Group (1 mentions)
Bovine serum albumins, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Dulbecco s modified eagle s medium dmem, by Sartorius (1 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Biotin peg3 azide, by Vector Laboratories (1 mentions)
Dadps biotin azide, by Vector Laboratories (1 mentions)
β lactoglobulin a, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Anti ki67 antibody, by Cell Signaling Technology (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Beyotime (1 mentions)
Biotin labeled secondary antibody, by Beyotime (1 mentions)
Sars cov 2 surrogate virus neutralization test kit, by GenScript (1 mentions)
14 protocols using hrp labeled streptavidin
1
Immunohistochemical Analysis of p53 in Kidney Tissues
2
Affinity Purification of Biotinylated Proteins
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The RHΔhxgprt-CaM-BirA* strain was used to infect HFF cells for about 24 h and then incubated 24 h with or without 150 μM biotin (Sigma-Aldrich, USA) in the D2 medium, as previously described (56 (link)). The parasite samples were collected by manual scraping, washed in PBS, and lysed in RIPA buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1% sodium deoxycholate; and 0.1% sodium dodecyl sulfate [SDS]; Beyotime, China) supplemented with protease inhibitor (ThermoFisher, USA) (36 (link)). The lysates were centrifuged at 12,000 × g for 10 min at 4°C. The supernatants were incubated for 6 h at 4°C with gentle tube rotation with MyOne Streptavidin magnetic beads T1 (ThermoFisher, USA). Magnetic beads were washed three times with RIPA buffer, then boiled for 10 min in 1× SDS-loading buffer to release biotinylated proteins. 10% of each sample was analyzed by Western blotting using HRP-labeled streptavidin (Beyotime, China), while the remaining samples were loaded in 12% SDS-PAGE gel and separated for about 10 min. The top gel containing biotinylated proteins was cut, freeze-dried, and used for LC-MS/MS.
3
Comprehensive Cysteine Labeling Protocol
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Iodoacetamide (IAA), glutathione (GSH), DL-dithiothreitol (DTT), tris(2-chloroethyl) phosphate (TCEP), copper sulfate (CuSO4), S-methyl methanethiosulfonate (MMTS) and calcium chloride (CaCl2) were purchased from Sigma-Aldrich unless otherwise specified. Fmoc-Cys and N-ethylmaleimide (NEM) was obtained from Heowns and J&K Scientific, respectively. Biotin-PEG3-azide, DADPS-Biotin-Azide and click chemistry auxiliary reagents (TBTA and BTTAA) was purchased from Click chemistry tools. Cy3-Azide and desthioBiotin-PEG3-azide was obtained from Okeanos and Confluore Biotechnology, respectively. Four peptides containing one cysteine, GCSWDYKN was synthesized from GL Biochem and the others were purchased from SciLight Biotechnology. All cell culture related reagents, such as fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM) and penicillin-streptomycin were purchased from Biological Industries (BI). Three kinds of proteins containing one free cysteine residues, β-lactoglobulin A, bovine serum albumins and papain were obtained from Sigma-Aldrich. All antibodies used for immunoblotting were purchased from Abcam except HRP-labeled Streptavidin (Beyotime Biotechnology), and other chemical or biological reagents were obtained from commercial suppliers without any manipulation.
4
Evaluating Tumor Cell Proliferation Inhibition
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To explore whether Poly Abs could effectively inhibit tumor cell proliferation in vivo, anti-Ki67 antibody (Cell Signaling, USA) was used to determine cellular proliferation in tumor tissues. Tumor tissue were fixed in 4 % paraformaldehyde 48 h latter last treatment, embedded in paraffin, and 4 µm sections were incubated with anti-Ki67 antibody (1:100) overnight at 4 °C. Sections were incubated with biotin-labeled secondary antibody (1:100, Beyotime, China) for 40 min, and then exposed to HRP-labeled streptavidin (1:200, Beyotime, China) for 20 min. Positive reactions were visualized using 3, 3'-diaminobenzidine tetrahydrochloride (DAB; Beyotime, China), followed by counterstaining with hematoxylin (Beyotime, China).
5
Quantifying IgA1, Aberrantly Glycosylated IgA1, and BAFF
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Culture supernatants were analyzed by specific ELISA for IgA1, aberrantly O-glycosylated IgA1, and BAFF. Mouse antihuman primary IgA1-specific antibody (Santa Cruz Biotechnology, USA) was fixed on the ELISA plate, followed by the sequential addition of human IgA1 protein standards (Calbiochem, USA), the samples, mouse biotin-labeled antihuman IgA-specific antibodies (Southern Biotech Associates, USA) for IgA1 while biotin-labeled lectin Vicia (Vector Laboratories Associates, USA) for aberrantly O-glycosylated IgA1, HRP-labeled Streptavidin (Beyotime Institute of Biotechnology, China), and TMB color liquid and stop solution (Beyotime Institute of Biotechnology, China). The optical density (OD) was measured at a wavelength of 450 nm by enzyme-linked immunosorbent assay to obtain concentrations of the supernatants. BAFF level was assessed by Human B-Cell-Activation Factor ELISA Kit (Cusabio, China), as per the manufacturer's recommendations.
6
IgA1 Quantification in Cell Supernatants
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The supernatant of the TMCs was collected and 96-well immunoplates (Costar, Cambridge, MA, USA) were coated with mouse anti-human IgA1 antibody (1:400, Santa Cruz Biotechnology) at 4°C overnight. Plates were washed five times with PBS containing 0.05% Tween-20, then blocked with PBS containing 1% BSA at 37°C for 2 h. Next, supernatant samples or standard human IgA1 (Calbiochem, La Jolla, CA., USA) were added to each well (100 µl) and incubated at 37°C for 2 h. The plates were washed five times, then biotin-labeled mouse anti-human IgA1 (1:2,000, Southern Biotechnology) was added to each well (100 µl) and incubated at 37°C for 1 h. The plates were washed five times, and HRP-labeled streptavidin (1:4,000, Beyotime Institute of Biotechnology) was added to each well (100 µl) and incubated at 37°C for 30 min. Finally, 0.1 mg/ml of tetramethylbenzidine (TMB) was added at room temperature for 5 min. The optical density (OD) was measured at 450 nm. Each sample was assayed in duplicate and repeated more than three times.
7
SARS-CoV-2 Spike Protein Binding ELISA
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ELISA was performed by a SARS-CoV-2 surrogate virus neutralization test kit (GenScript catalog no. L00847), which contained an hACE2-precoated microplate. Briefly, 0.1 μg/well of SARS-CoV-2 spike protein with a streptavidin tag or the horseradish peroxidase (HRP)-conjugated recombinant SARS-CoV-2 RBD fragment was incubated with ATRA at 37°C for 30 min. DMSO was used as a negative control, and the hACE2 antibody was used as a positive control. The mixture was then added to the precoated microplate at 37°C for 15 min, and the unbound protein was removed by three washes with the wash buffer provided with the kit. For the spike binding ELISA, HRP-labeled streptavidin (Beyotime catalog no. A0303) was added at a dilution of 1:4,000, and the mixture was incubated at 37°C for 45 min. After four washing steps, TMB (3,3′,5,5′-tetramethylbenzidine) solution was added to the microplate, and the mixture was incubated at room temperature. Then, the stop solution was added, and the absorbance was detected at 450 nm.
8
Naringin Modulates NF-κB p65 in Rat Retina
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IHC assay was performed to detect the effects of naringin on expression of NF-κB p65 in rat retinal tissues. Sample sections (5 μm) were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed with sodium citrate buffer in a microwave for 10 min. The sections were added with 3 % H2O2 for 15 min at room temperature (RT). Thereafter, the sections were blocked with goat serum (Solarbio) for 15 min at RT. After serum deprivation, the sections were incubated with NF-κB p65 primary antibody (1:50; Santa cruz, Santa Cruz, CA, USA) at 4 °C overnight and then with a Biotin-labeled Goat Anti-Rabbit IgG(H+L) secondary antibody (1:200; Beyotime, Beijing, China) at 37 °C for 30 min. Then the sections were incubated with HRP-labeled streptavidin (Beyotime) at 37 °C for 30 min and color was developed using DAB (Solarbio). The sections were counterstained with hematoxylin (Solarbio). The positive staining was visualized using a microscope (Olympus).
9
Immunohistochemical Analysis of CARD10 and β-Catenin
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The tumor nodules from mice were subsequently removed and fixed into paraffin sections. The fixed tissue paraffin sections were first incubated in 3% hydrogen peroxide for 15 mins at RT. After blocking, they were incubated with anti-CARD10 (1:200, bs-7081R, Bioss, China) or anti-β-Catenin (1:100, #8480, Cell Signaling Technology, USA) at 4 °C overnight, and then incubated in Biotin-labeled Goat Anti-Rabbit IgG (H + L) (1:200, A0277, Beyotime, China) at 37 °C for 30 mins. Thereafter, the protein signals were magnified with HRP-labeled Streptavidin (1:200, A0303, Beyotime, China), and visualized with DAB (DA1010, Solarbio, China). Cell nuclei were counterstained with hematoxylin (H8070, Solarbio, Beijing, China). Images were captured under a microscope.
10
Immunohistochemistry and Flow Cytometry Protocols
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The primary antibodies used for immunohistochemical staining were purchased from R&D Systems (Minneapolis, MN, USA): anti-C3d; Bethyl Laboratories (TX, USA): anti-IgG; the secondary antibodies were purchased from Beyotime Biotechnology (Shanghai, China): HRP-labeled anti-anti-C3d and HRP-labeled streptavidin. The antibodies used for flow cytometric analysis were purchased from BioLegend (San Diego, CA, USA): BV570-CD45, FITC-CD4, APC-F4/80, BV421-B220; eBioscience (San Diego, CA, USA): eFluor 450-CD8a, Alexa Fluor 700-CD3, PE-CD49b; BD Biosciences: PE-Cy7-CD11b; Abcam (Cambridge, England): anti-IgG; and Jackson ImmunoResearch Laboratories (West Grove, PA, USA): anti-IgM.
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