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Histone H3 (96C10) Mouse mAb (IHC Formulated) #3680
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Histone H3 (96C10) Mouse mAb (IHC Formulated).Learn more about how we got this image
Gallery: Histone H3 (96C10) Mouse mAb (IHC Formulated) #3680
A. Solutions and Reagents
- Ethanol, anhydrous denatured, histological grade (100% and 95%)
- Deionized water (dH2O)
- Hematoxylin (optional)
- Wash Buffer:
- 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
- SignalStain® Antibody Diluent (#8112).
- Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7â¢2H2O) to 1 L dH2O. Adjust pH to 6.0.
- 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
- Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
- Substrate: Vector® NovaRED™ (Vector Laboratories).
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 minutes each.
- Incubate sections in two washes of 100% ethanol for 10 minutes each.
- Incubate sections in two washes of 95% ethanol for 10 minutes each.
- Wash sections twice in dH2O for 5 minutes each.
C. Antigen Unmasking
For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
- Wash sections in dH2O three times for 5 minutes each.
- Incubate sections in 3% hydrogen peroxide for 10 minutes.
- Wash sections in dH2O twice for 5 minutes each.
- Wash sections in wash buffer for 5 minutes.
- Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
- Remove blocking solution and add 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Prepare ABC solution per manufacturer's recommendations.
- Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
- Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
- Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
- Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
- Wash section three times with wash buffer for 5 min each.
- Prepare Vector® NovaRED™ per manufacturer's recommendations.
- Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
- If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 minutes each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 seconds each.
- Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
- Repeat in xylene, incubating sections two times for 10 seconds each.
- Mount coverslips.
posted June 2005
revised February 2008
protocol id: 293
Histone H3 (96C10) Mouse mAb (IHC Formulated) detects endogenous levels of total histone H3 protein. The antibody does not cross-react with other histone proteins.Species Reactivity: Human Species predicted to react based on 100% sequence homology: Mouse, Rat, Monkey, D. melanogaster, Xenopus, Horse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of human histone H3.
Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11).
Protein Specific References
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.