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SY SY - Synaptic Systems SY SY - Synaptic Systems

Protocol for immunohistochemistry - paraffin sections

Solutions needed:

  • Xylene

  • Ethanol

  • 10 mM citrate buffer: pH 2.5-6.0 (varies from antibody to antibody)

  • TBS: 20 mM Tris, pH 7.3 / 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

Deparaffinization and rehydration:

  1. Incubate sections with xylene twice for 5 min.

  2. Rehydrate sections through a series of ethanol concentration ranging from 100 % to 70 % (10 % steps) with a minimum of 10 min/concentration.

  3. Rinse sections in TBS.

Antigen retrieval:

  1. Water bath sections in 10 mM citrate buffer at 95°C for 40 min to retrieve antigens that are masked through the embedding process.

  2. Cool slides slowly to RT.

  3. Rinse slides in TBS.

Immunofluorescence:

  1. Block in blocking solution for 30 min at RT.

  2. Remove blocking solution and incubate sections overnight at 4°C with the primary antibody diluted in blocking solution (optimal dilution must be determined experimentally).

  3. Remove primary antibody and wash thoroughly with TBS three times for 10 min.

  4. Incubate with the secondary antibody diluted in incubation solution for 1 h at RT. Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye.

  5. Remove secondary antibody and wash thoroughly with TBS three times for 10 min.

  6. Mount slices 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