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SY SY - Synaptic Systems SY SY - Synaptic Systems

Protocol for immunohistochemistry - cryo 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

  • 20 % sucrose in 0.1 M Tris-HCl, pH 7.2

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

  • 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 with 0.9 % saline for 1 min.

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

  3. Fix tissue in fixation solution overnight.

  4. Rinse tissue in 0.1 M Tris-HCl, pH 7.2 and change buffer three times.

  5. Incubate tissue for 12-18 h in 20 % sucrose, 0.1 M Tris-HCl, pH 7.2.

  6. Freeze tissue with TISSUE-TEK using a cryostat.

  7. Cut tissue (6-10 ) and mount sections on Super Frost Ultra Plus® slides.

  8. Allow frozen sections to dry for 15 min at RT.

  9. Block for 1-2 h in blocking solution.

  10. Incubate with primary antibody diluted in incubation buffer overnight at 4C (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 1 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.




Synaptic Systems Gesellschaft für neurobiologische Forschung, Entwicklung und Produktion mbH | Rudolf-Wissell-Str. 28 | 37079 Goettingen | Germany
Phone: +49 (0)551/505 56-0 | Internet: http://www.sysy.com | E-Mail: sales@sysy.com | Imprint
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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