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Standard protocol for immunocytochemistry of methanol fixed cultured neurons

This protocol is recommended for some proteins of the post-synaptic density (PSD)

Methanol fixation works by denaturing and precipitating proteins, and as such it is a quick method. For most antibodies/proteins it takes only 2 - 5 minutes. This procedure leads to an unmasking of the proteins in the PSD.

Solutions needed:

  • MES: 100 mM MES, pH 6.9; 1 mM EGTA; 1 mM MgCl2. Store at 4°C

  • Methanol fix (100 ml): 10 ml MES; 90 ml methanol. Store at -20°C

  • PBS, pH 7.4 (0.1 % Tween-20 can be added for the washing steps).

  • 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 in PBS (normal serum from the host-species of the secondary antibodies is recommended for blocking).

Procedure:

  1. Pour some -20°C methanol fix carefully onto the plated cells and leave the dish on the bench for 5 min.

  2. Wash three times with PBS for 10 min.

  3. Incubate for 30 min with blocking solution.

  4. Put a piece of parafilm on wet Whatman paper and apply 200 µl of primary antibody in incubation solution (optimal dilution must be determined experimentally).

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

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

  7. Repeat steps 4-6 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.

  8. Wash three times with PBS for 10 min.

  9. Add a drop of mounting medium on slide and mount coverslip.

  10. Incubate for 30 min at RT.

  11. Incubate over night at 4°C.




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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