Laboratory Protocols

Peroxidase-anti-peroxidase Technique

For The Identification Of Antigens In Paraffin Wax Sections

(Use slide coated with POLY-L-LYSINE to prevent section loss).

  1. Dewax sections and transfer to 100% alcohol.
  2. Block endogenous peroxidase with 0.5% hydrogen peroxide in methanol - 30 minutes.
  3. Place in tris-saline buffer at 20oC.
  4. Treat with 0.05% trypsin in tris-saline buffer containing 0.1% calcium chloride at 37oC (must be accurate). Add the trypsin to the rest after preheating and just before use - 40 minutes.
  5. Wash in several changes of tris-saline at 20oC.
  6. Treat with P.B.S. containing 1% normal serum (P.B.S./N.S.) from the species the secondary was raised in - 2 x 5 minutes.
  7. Treat with specific primary antibody diluted to the order of 1:100 to 1:250 for immunoglobulins or 1:250 to 1:500 for hormones with P.B.S./N.S. - 30 minutes in a damp chamber.It may be necessary to extend this period of incubation to 12-24 hours.
  8. Wash in P.B.S./N.S. - 2 x 5 minutes.
  9. Treat with secondary antibody (anti-(primary host)-IgG) diluted 1:40 with P.B.S. - 20 minutes.
  10. Wash in P.B.S./N.S. - 2 x 5 minutes.
  11. Treat with P.A.P. (from same species as the primary) diluted 1:40 with P.B.S. - 20 minutes.
  12. Wash in P.B.S./N.S. - 2 x 5 minutes.
  13. Rinse in 0.05M pH5 acetate buffer for ethylcarbazole; T.B.S. for D.A.B.
  14. Treat with peroxidase substrate solution...ethylcarbazole or D.A.B. - 5 minutes.
  15. Rinse in distilled water.
  16. Stain in Mayer's haematoxylin - 30 seconds.
  17. Wash and blue in running water.
  18. Mount in glycerine jelly or a suitable aqueous mountant. (For D.A.B. dehydrate in alcohol, clear in xylene or Histo-Clear® and mount in D.P.X.).

Controls

  1. Omit the primary antibody by leaving grids in the wash/block solution at step 6. and continuing to step 8. (Checks the secondary and substrate).
  2. Replace the primary antibody with another but inappropriate antibody. (Checks the primary).
  3. Replace the primary antibody with normal (non-immune) serum obtained from the same animal as the primary. (Checks the primary).

Solutions

Tris-saline buffer (pH 7.8)

Tris 0.2M in distilled water - 10 parts.

0.1M HCl - 13 parts.

Physiological saline (0.85%) - 17 parts.

Trypsin solution

5% trypsin - 8mls.

5% calcium chloride - 16mls.

Tris-saline buffer - 776mls.

0.05M acetate buffer

0.1M acetic acid (2.85mls glacial in 500mls distilled water) - 50mls.

0.1M sodium acetate (4.1g anhydrous or 6.8g trihydrate in 500mls distilled water) - 150mls.

Distilled water - 200mls.

Ethylcarbazole reagent

0.002g 3-amino-ethylcarbazole dissolved in 0.5mls N,N,dimethylformamide in a DRY glass container. Add 9.5mls pH5 0.05M acetate buffer and use immediately.

D.A.B. reagent

3,3 diaminobenzidine tetrahydrochloride - 6mg.

Tris-saline buffer - 10mls.

3% hydrogen peroxide - 0.1mls.

0.01M phosphate buffered saline

NaCl - 17g. Na2HPO4 (MW 142) - 2.67g.

NaH2PO4 (MW 156) - 0.41g.

Make up to 2 litres with distilled water.