The specimens were fixed for one week in 10% formaldehyde, decalcified for four weeks in 0.05% EDTA, and then embedded in paraffin. Serial sagittal sections of IVDs (5 μm thickness) were prepared, deparaffinized in dimethylbenzene, and rehydrated. Some sections were stained with hematoxylin and eosin (H&E) and Alcian Blue to evaluate the severity of degeneration. For immunohistochemical staining, other sections were incubated with anti SDF-1 (1 : 100 dilution; Cell Signaling Technology, MA, USA) overnight at 4°C. On the second day, all sections were washed with PBS and were incubated with the secondary antibody conjugated with horseradish peroxidase (1 : 200 dilution; Abcam, MA, USA) at 37°C for 1 h, followed by color development with diaminobenzidine (DAB, Beyotime). Then the sections were counterstained with hematoxylin (Beyotime). The stained sections were examined using a light microscope (Nikon).
Secondary antibody conjugated with horseradish peroxidase
Manufactured by Abcam
Sourced in United States
Secondary antibody conjugated with horseradish peroxidase. This product is a detection reagent used in immunoassays and immunohistochemistry to amplify and visualize the signal from a primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Beyotime (1 mentions)
Diaminobenzidine dab, by Beyotime (1 mentions)
Anti sdf 1, by Cell Signaling Technology (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Westernbright ecl kit, by Advansta (1 mentions)
Anti fam134b, by Abcam (1 mentions)
Anti bnip3, by Abcam (1 mentions)
Anti ip3r, by Abcam (1 mentions)
Anti facl4, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Anti calnexin, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti β actin, by Abcam (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Anti cytochrome c, by Abcam (1 mentions)
3 protocols using secondary antibody conjugated with horseradish peroxidase
1
Histological and Immunohistochemical Analysis of IVDs
2
Knockdown of EIF4EBP1 in Tissue Samples
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Short interference RNAs (siRNAs) for EIF4EBP1 and corresponding scrambled siRNA negative controls were synthesized by GenePharma (Shanghai, China). The EIF4EBP1 plasmid and negative control vector were purchased from GenePharma (Shanghai, China). Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) was used to transiently transfect cells according to the manufacturer’s instructions. The transfection efficiency was evaluated by RT-qPCR after 24 h. Then, the slides with tissue samples were heated in sodium citrate buffer (95 °C, 10 min) and sealed in normal goat serum (37 °C, 1 h). The tissue samples were incubated with an anti-EIF4EBP1 antibody (Abcam, Shanghai, China) for 1 h at 37 °C. Then, the tissue samples were incubated with a secondary antibody conjugated with horseradish peroxidase (Abcam). The experimental results were independently assessed by three blinded pathologists and analyzed using ImageProPlus software (version 6; MediaCybernetics, Rockville, MD, United States).
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from frozen placental tissues and HTR8/ SVneo cells with RIPA lysis buffer (Beyotime, Shanghai, China). Following the manufacturer's protocol, the protein concentration was determined using a BCA Protein Assay kit (Beyotime). Western blot analysis was performed based on the technique established in our laboratory [26] . As previously described, protein samples were subjected to SDS polyacrylamide gel electrophoresis, resolved by electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA) [27] (link). The antibodies used were anti-FAM134B (1:1000 dilution; Abcam), anti-IP3R (1:1000 dilution; Abcam), anti-FACL4 (1:1000 dilution; Abcam), anti-cytochrome C (1:500 dilution; Abcam), anti-cleaved caspase-3 (1:1000 dilution; Abcam), anti-IP3R (1:1000 dilution; Abcam), anti-calnexin (1:1000 dilution; Abcam), anti-BNIP3 (1:1000 dilution; Abcam), anti-PSME2 (1:1000 dilution; Abcam), and anti-β-actin (1:1000 dilution; Abcam). All incubation was performed at 4°C overnight. The polyvinylidene difluoride membranes were then incubated with a secondary antibody conjugated with horseradish peroxidase (1:5000 dilution; Abcam). The protein bands were visualized using western bright ECL kit (Advansta, Menlo Park, USA) and quantified with the ChemiDoc™ XRS+(Bio-Rad, Hercules, USA).
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