PMN Rabbit Polyclonal Antibody
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Applications | CT, IHC |
Recommended Dilution | Immunohistochemistry on frozen and paraffin sections. Cytotoxic assays. For Cytotoxic Antibodies: Modified Colormetric Microtiter Assay 1 Results: Antisera of this antibody diluted 1/50 exhibits 80% cytotoxicity on mouse PMN. Antisera of this antibody diluted 1/50 exhibits <5% cytotoxicity on mouse thymocytes or splenocytes. For Agglutinating Antibodies: Antisera dilutions in RPMI-1640 incubated with target cells at 4°C-8°C for 1hr. Agglutination determined by microscopic observations. Results: Antisera of this antibody strongly agglutinates mouse PMN but not thymocytes at dilutions to 1/100. |
Reactivities | Mouse |
Host | Rabbit |
Clonality | Polyclonal |
Specificity | This antibody reacts to PMN. |
Formulation | State: Serum State: Liquid serum |
Synonyms | Polymorphonuclear Leukocytes |
Note | Protocol: Rabbit anti-Mouse PMN IHC protocol Sections: Animals were perfused with 10 mM PBS, pH 7.4 followed by 4% paraformaldehyde in 100 mM phosphate buffer, pH 7.4. Tissue was removed and placed in 4% paraformaldehyde for 6 hours at room temperature, then transferred to 10 mM PBS, pH 7.4 in 0.15M isotonic saline at 4°C overnight. Sections were cut at 50 μm on a vibratome using 10 mM PBS and placed in tissue culture wells at 4°C overnight. Procedure: 1. Pretreat slices in 50 mM NH4Cl (0.267g/100 ml PBS) in PBS at 22°C for 1 hour. 2. Pretreat slices in 0.1% Triton X-100 in PBS at 22°C for 1 hour. 3. Wash in PBS for 5 minutes at 22°C. 4. Block in PBS with 5% NGS at 22°C for 2 hours. 5. Incubate in primary antibody (Rabbit anti-mouse PMN), AP31660SU-N, diluted 1/3000 in PBS with 5% NGS overnight at 4°C. 6. Wash in PBS with 5% NGS at 22°C for 30 minutes. 7. Incubate in secondary antibody, biotinylated goat anti-rabbit IgG in PBS with 5% NGS at 22°C for 1 hour, diluted: 1) 1/200, 125 μl/25 ml 2) Per instructions, Vector ABC Elite Kit, 8 drops/25 ml 8. Wash in PBS with 5% NGS at 22°C for 30 minutes. 9. Block endogenous peroxidase activity. Immediately before use, mix 81 ml PBS, 9 ml methanol and 10 ml 30% H2O2. Incubate for 10 minutes at 22°C. 10. Wash in PBS only 1 x 10 minutes at 22°C. 11. Wash in PBS only 1 x 20 minutes at 22°C 12. Wash in PBS with 5% NGS at 22°C for 30 minutes. Prepare ABC reagent, if using this option. 13. Incubate in: 1) KPL Streptavidin-peroxidase conjugate diluted 1/200 (125 μl/25 ml) in PBS with 5% NGS, 0.1% Tween 20. 2) Vector ABC elite, diluted according to kit instructions for 1 hour at 22°C. 14. Wash in PBS with 5% NGS at 22°C for 10 minutes. 15. Incubate with DAB (2-5 minutes). Stop reaction with PBS wash. 16. Allow to air dry. 17. Wash salts off in ddH2O. Dehydrate, clear and mount on chromalum gelatin coated slides. Notes: Paraformaldehyde: 2.76g monobasic 21.45g dibasic heptahydrate 1000 ml ddH2O 40g Paraformaldehyde PBS (10mM): 0.276 monobasic 2.15g dibasic heptahydrate 100 ml ddH2O 8.76 NaCl/1000 ml 900 ml Isotonic saline + 100 ml mono/dibasic pH to 7.4 Primary antibody: 17 μl/50 ml PBS with 5% NGS |
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