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SY SY - Synaptic Systems SY SY - Synaptic Systems

Standard protocol for immunocytochemistry of PFA fixed cultured neurons

Solutions needed:

  • Phosphate buffer saline (PBS), pH 7.4

  • 4 % Paraformaldehyd in PBS, pH 7.4 / 4 % sucrose

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

  • Incubation solution: 3 % normal serum / 0.1 % Triton X-100/ PBS (Triton may be omitted; normal serum from the host-species of the secondary antibodies is recommended for blocking).

  • Mounting medium

Procedure:

  1. Wash coverslips twice with PBS.

  2. Fix cells with 3 ml/well paraformaldehyde solution for 15-20 min at RT.

  3. Wash three times with PBS for 10 min.

  4. Incubate for 30 min with blocking solution.

  5. Put a piece of parafilm on wet Whatman paper and apply 200 ┬Ál of primary antibody solution in incubation solution (appropriate dilution must be determined experimentally).

  6. Put coverslips upside down on antibody-solution and incubate for 2 h at RT.

  7. Transfer slips to multi-well plate and wash three times with PBS for 10 min.

  8. Repeat steps 5-7 with secondary antibody (optimal dilution must be determined experimentally). Avoid bright light when working with the secondary antibody to minimize photo bleeching of the fluorescent dye.

  9. Mount coverslips on slides 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