PMN Rabbit Polyclonal Antibody

Specifications

Product Data
Applications	CT, IHC
Recommend Dilution	Cytotoxic assasy. Immunohistochemistry on frozen and paraffin sections. For Cytotoxic Antibodies: Modified Colorimetric Microtiter Assay 1 Results: Antisera of the above this antibody diluted 1/20 exhibits >80% cytotoxicity on rat PMNs Antisera diluted 1/20 exhibits <15% cytotoxicity on rat 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 the above this antibody strongly agglutinates rat PMNs but not lymphocytes at dilutions 1/20 to 1/200
Reactivity	Rat
Host	Rabbit
Clonality	Polyclonal
Specificity	This antibody reacts to PMN.
Formulation	State: Serum State: Liquid serum
Conjugation	Unconjugated
Storage Condition	Upon receipt, store (in aliquots) at -20°C to -80°C. Avoid repeated freezing and thawing.
Background	CLAD51140 is an antiserum directed against rat PMNs (Polymorphonuclear Leukocytes). This antisera is adsorbed repeatedly with red blood cells and lymphocytes to remove most antibodies to antigens common among different cell types. This adsorption significantly improves cytotoxic specificity.
Synonyms	Polymorphonuclear Leukocytes
Note	Protocol: Rabbit anti-Rat PMN IHC protocol (can also be used for mouse sections) 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-rat PMN), AP31661SU-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
Reference Data

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