PMN Rabbit Polyclonal Antibody

CAT#: AP31660SU-N

PMN rabbit polyclonal antibody, Serum

Specifications

Product Data

• 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
• Conjugation: Unconjugated
• Storage: Store the antibody at -20°C.
  Avoid repeated freezing and thawing.
• Stability: Shelf life: one year from despatch.
• 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