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SY SY - Synaptic Systems SY SY - Synaptic Systems

Protocol for immunohistochemistry - Alzheimer's disease plaques (vibratome sections)

Solutions needed:

  • 0. 9% saline

  • Fixation solution: 4 % paraformaldehyde / 50 mM phosphate buffer, pH 7.2 / 150 mM NaCl.

  • 0.1 M Tris-HCl, pH 7.2

  • TBS: 20 mM Tris, pH 7.2 / 150 mM NaCl

  • Formic acid (optional)

  • Blocking solution: 10 % normal serum / 0.3 % Triton-X 100 in TBS (normal serum from the host-species of the secondary antibodies is recommended for blocking).

  • Incubation buffer: 5 % normal serum / 0.3 % 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 0.9 % saline for 1 min.

  2. Perfuse with fixation solution for 10-20 min with a rate of 12 ml/min.

  3. Incubate tissue in fixation solution overnight at 4°C.

  4. Wash tissue in 0.1 M Tris-HCl, pH 7.2 for 10 min.

  5. Cut tissue (40-60 µ) with a vibratome.

  6. Wash twice with 0.1 M Tris-HCl, pH 7.2 for 10 min.

  7. Treat sections with formic acid for 10 min at RT (optional).

  8. Wash twice with TBS for 10 min.

  9. Block for 1 h in blocking solution.

  10. Incubate with primary antibody diluted in incubation buffer overnight at 4°C (optimal dilution must be determined experimentally).

  11. Wash three times for 10 min in TBS.

  12. 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.

  13. Wash three times in TBS for 10 min.

  14. Mount with 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