Mouse hippocampal tissues on P7 were harvested and lysed in cell lysis buffer (Cell Signaling, MA) containing phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN) for protein preparation. Total protein of 10 micrograms (μg) was loaded for Western blot assay. The primary antibodies used were rabbit anti-cleaved caspase 3 (Cell Signaling, Danvers, MA), and rabbit anti-actin (Cell Signaling, Danvers, MA). The secondary antibody used was anti-rabbit immunoglobulin G horseradish peroxidase-linked (Cell Signaling, Danvers, MA). The protein membranes were developed using an ECL Detection kit (GE Healthcare Life Sciences, Marlborough, MA) and signals were captured using a Chemidoc imaging system (Bio-Rad, Hercules, CA). Actin was used as a loading control during protein normalization of cleaved caspase 3.
Anti rabbit immunoglobulin g horseradish peroxidase linked
Manufactured by Cell Signaling Technology
Anti-rabbit immunoglobulin G horseradish peroxidase-linked is a secondary antibody used in immunodetection techniques. It binds to rabbit primary antibodies and is conjugated with the enzyme horseradish peroxidase, which can be detected using a suitable substrate.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Rabbit anti actin, by Cell Signaling Technology (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
β actin antibody clone ac 15, by Merck Group (1 mentions)
Gapdh antibody 6c5, by Abcam (1 mentions)
Brca1, by Santa Cruz Biotechnology (1 mentions)
Rnf168, by Merck Group (1 mentions)
Alexa fluor 594 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
γh2ax, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Flag m2, by Merck Group (1 mentions)
Mts reagent kit, by Promega (1 mentions)
4 protocols using anti rabbit immunoglobulin g horseradish peroxidase linked
1
Quantifying Apoptosis in Mouse Hippocampus
2
Protein Extraction and Western Blot Analysis
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Frozen liver tissue samples or cultured cells were homogenized in lysis buffer (10 mM Tris/HCl pH 7.6, 5 mM ethylene diamine tetraacetic acid, 50 mM NaCl, 1% Triton X‐100, complete protease inhibitor cocktail, and 50 mM NaF) and centrifuged (10,000g, 20 minutes, 4°C). Protein concentration was determined by using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). After quantification, 10‐25 μg of protein was electrophoresed on sodium dodecyl sulfate‐polyacrylamide gels and transferred onto membranes. Membranes were incubated with the following antibodies: SCD1 (C12H5) rabbit monoclonal antibody (2794S; Cell Signaling Technology, Danvers, MA), glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody [6C5] (ab8245; Abcam, Cambridge, United Kingdom), β‐ACTIN antibody clone AC‐15 (A5441; Sigma‐Aldrich, St. Louis, MO), and COLLAGEN I type I antibody [C‐18] (sc‐8784; Santa Cruz Biotechnology, Dallas, TX). As secondary antibodies, we used anti‐rabbit immunoglobulin G‐horseradish peroxidase‐linked and anti‐mouse immunoglobulin G‐horseradish peroxidase‐linked (Cell Signaling Technology) antibodies. Densities were analyzed by Image J (National Institutes of Health, Bethesda, MD) software. For densitometric quantification, levels of each protein for each sample were normalized to loading controls (GAPDH or β‐ACTIN).
3
Antibody Panel for DNA Damage Response
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Primary antibodies used in this study were UBA80 (Raybiotech, 144-02027-50), UBA52 (Bio-Rad, VPA00424), Flag M2 (Sigma, F1804), Myc (Santa Cruz, sc-40), GFP (Invitrogen, A11122 ), 53BP1 (Novus Biologicals, NB100-304), BRCA1 (Santa Cruz Biotechnology, SC-6954), γH2AX (Millipore, 05-636), H2AX (Cell Signaling, 2595S), H2A (Cell Signaling, 2578), tubulin (Abcam, ab6046), MDC1 (Abcam, ab11169), RNF168 (Sigma-Aldrich, ABE367), GST (Millipore, 71007-3), and MBP (Abcam, ab119994). For Western blotting, secondary antibodies—horseradish peroxidase-linked anti-rabbit immunoglobulin G and horseradish peroxidase-linked anti-mouse immunoglobulin G—were purchased from Cell Signaling (0704 and 0706). For immunofluorescence, Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 594 goat anti-mouse antibodies were used (Invitrogen).
4
Western Blot Analysis of NR2B in CA3 Subregion
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Tissue from the CA3 subregion was isolated for assaying. Tissues and cells were lysed by radioimmunoprecipitation assay lysis buffer, and total protein was extracted for western blotting experiments. The primary antibodies were NR2B (Cell Signaling Technology, 14544s) and glyceraldehyde phosphate dehydrogenase (Cell Signaling Technology, 5174s), and the secondary antibody was horseradish peroxidase-linked anti-rabbit immunoglobulin G (Cell Signaling Technology, 7074s). After 24 h of irradiation, the cell activity was detected using the MTS reagent kit (Promega, G3581).
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