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SY SY - Synaptic Systems SY SY - Synaptic Systems

Protocol for immunohistochemistry - methanol/acetic acid treatment

Solutions needed:

  • 0. 9% saline

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

  • Phosphate buffer: 100 mM phosphate buffer, pH 7.3

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

  • 20 % sucrose in 0.1 M Tris-HCl, pH 7.3

  • 95 % methanol / 5 % acetic acid solution, keep at -20°C

  • Blocking solution: 10 % normal serum / 0.1 % Triton-X 100 in TBS (normal serum from the host-species of the secondary antibodies is recommended for blocking).

  • Incubation buffer: 2 % normal serum / 0.1 % Triton-X 100 in TBS (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. Wash with phosphate buffer for 10 min.

  4. Incubate tissue for 12-18 h in 20 % sucrose, 0.1 M Tris-HCl, pH 7.3.

  5. Wash with phosphate buffer for 10 min.

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

  7. Wash twice with phosphate buffer for 10 min.

  8. Treat sections with ice cold methanol/acetic acid solution for 10 min.

  9. Wash in TBS for 10 min.

  10. Block for 1 h in blocking solution.

  11. Incubate with primary antibody diluted in incubation buffer overnight at RT (optimal dilution must be determined experimentally).

  12. Wash three times for 10 min in TBS.

  13. 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.

  14. Wash three times in TBS for 10 min.

  15. Mount with fluorescent mounting medium 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