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Immunofluorescence on ultrathin resin sections

来源:生物谷  2010/5/16   访问量:2585
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General remarks:
Ultrathin methacrylate-(e.g.Lowicryl HM20, K4M, Monostep polar and nonpolar or LR-White) or epoxy (Epon/Araldite, Spurr) sections are transferred with a loop on poly-L-lysine coated, round cover slips. Residual water is drained with filter paper along the outside of the loop.

Note:
air-dried resin sections can be stored for months prior to labelling.

Procedure:
  • Encircle sections with a water repellent silicon pen (e.g. PAP-Pen from Electron Microscopy Sciences, Ft. Washington, PA or Sciences Services, Munich).
  • Incubate sections with blocking buffer, e.g. PBG (0.2 gelatin, 0.5 BSA in PBS or TRIS) or 1 milk powder in PBS for 10 min.
  • Remove blocking buffer, add 25 µl of the primary antibody solution per coverslip (with a final concentration in the range of 1-5 µg specific IgG/ml) and incubate for 30 -60 min.
  • Wash 5 times with buffer and incubate with fluorochrome-labelled second antibodies, similar to the primary antibody staining conditions. s Wash 5 times with buffer and counterstain nuclei with DAPI, Hoechst dye or propidium iodide (0.4-0.1 µg/ml in H2O) for 5 min.
  • After a final wash with buffer mount coverslips on glass slides using a small drop of mounting medium (like Elvanol or Moviol 4.88) for semipermanent embedding. The addition of anti-fading agents like DABCO (25-100 mg/ml), Paraphenylenediamine (1 mg/ml) or n-propyl gallate (10 mg/ml) is strongly recommended.
  • Use oil immersion objectives to examine.
    CAVEAT: All solutions should be centrifuged before use for 2 min. at 10,000 rpm. Avoid air-drying during all incubation steps.

References:

Immunofluorescence on ultrathin resin sections:

  • Albrecht U, Seulberger H, Schwarz H, and Risau W. (1990) Brain Res.535, 49-61.
  • Schwarz H, Hohenberg H, and Humbel BM. (1993) Immuno-Gold Electron Microscopy in Virus Diagnosis and Research (CRC Press, Boca Raton), pp 349-376.
  • Schwarz H, Müller-Schmid A, and Hoffmann W. (1993) Cell Tissue Res. 273, 417-425.
  • Schwarz H. (1994) Electron Microscopy 1994 ICEM 13-Paris (Jouffrey B, and Colliex C, eds.), Les Editions de Physique, Les Ulis, France, Vol. 3, 255-256.
  • Fialka I, Schwarz H, Reichmann E, Busslinger M, and Beug H. (1996) J. Cell Biol. 132, 1115-1132.
  • Kurth T, Schwarz H, Schneider S, and Hausen P. (1996) Cell Tissue Res. 286, 1-12.

Semi-permanent mounting medium containing polyvinylalcohols (Elvanol/Moviol):

  • Rodriguez J, and Deinhardt F. (1960) Virology 12, 316.
  • Lennette OA. (1978) Am. J. Clin. Path. 69, 647-648.

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