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SY SY - Synaptic Systems SY SY - Synaptic Systems

Protocol for immunohistochemistry - pepsin treatment

Solutions needed:

  • 0. 9% saline

  • Fixation solution:4 % paraformaldehyde / 100 mM phosphate buffer, pH 7.3 / 15 % v/v picric acid.

  • Phosphate buffer: 100 mM phosphate buffer, pH 7.3

  • TBS: 20 mM Tris, pH 7.3 / 150 mM NaCl

  • Pepsin solution: 0.2 mg/ml pepsin in 0.2 M HCl (IHC grade)

  • Blocking solution: 10 % normal serum / 20 mM Tris, pH 7.3 / 150 mM NaCl (normal serum from the host-species of the secondary antibodies is recommended for blocking).

  • Incubation buffer: 2 % normal serum / 0.1 % Triton-X 100 / 20 mM Tris, pH 7.3 / 150 mM NaCl (normal serum from the host-species of the secondary antibodies is recommended for blocking).

  • Mounting medium

Procedure:

  1. Transcardially perfuse with 0.9 % saline for 1 min.

  2. Perfuse with fixation solution for 10-15 min with a rate of 12 ml/min.

  3. Cut 60 µm thick coronal sections with a vibratome.

  4. Wash twice with phosphate buffer for 10 min.

  5. Treat sections with pepsin solution for 10-20 min.

  6. Wash in TBS for 10 min.

  7. Block for 1 h in blocking solution.

  8. Incubate with primary antibody diluted in incubation buffer overnight at RT.

  9. Wash three times in TBS for 10 min.

  10. Incubate with secondary antibody diluted in incubation buffer for 2 h at RT. Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye.

  11. Wash three times in TBS for 10 min.

  12. Mount with fluorescent mounting medium.

  13. Incubate for 30 min at RT.

  14. Incubate overnight at 4°C.

  15. Incubate for 30 min at RT.

  16. Seal with nail polish and microscope.




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Synaptic Systems GmbH | Rudolf-Wissell-Str. 28 | 37079 Göttingen | Germany
Phone: +49 (0)551/505 56-0 | Internet: http://www.sysy.com | E-Mail: sales@sysy.com