Staining Protocols for Lymphoid Tissue:

Technovit Glycol Methacrylate: Technical Information and Help From Heraeus Kulzer.

Normal immunostaining procedures for routine markers on sections obtained from lymphoid tissue.

Procedure:

  • Dry the sections for 2 hours at 37C on a slide warmer. This must be done whether the slides have been stored at 4C for some time or just collected.
  • Pretreat the sections with trypsin at 37C. Trypsin concentrations must be determined separately for each antibody. Use trypsin solution that has been preheated at 37C for 30 minutes. Cover section with at least 100 microliters of solution.
  • Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
  • Preincubate in normal serum from the animal species in which the second antibody is raised for 30 minutes at 37C. Only perform this step if aspecific background is present using a particular antibody.
  • Drip off excess serum and apply the first antibody in an appropriate concentration and incubate for 2 hours at 37C.
  • Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
  • Block endogenous peroxidase in a solution of 0.06% hydrogen peroxide in phosphate buffered saline, pH 7.4, for 30 minutes at room temperature.
  • Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
  • Incubate in appropriate dilutions of the secondary antibody, containing 5% normal serum for 60 minutes at room temperature.
  • Wash in PBS for 10 minutes at room temperature. Refresh the buffer 4 to 5 times.
  • Develop the peroxidase activity in daminobenzidine (DAB).
  • Counterstain the sections in hematoxylin or, if a more advanced morphological detail is necessary, in periodic-acid-Schiff reagent.
  • Cover with glycerin-gelatin and cover glass

     


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