Immunofluorescence on ultrathin resin sections
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Immunofluorescence on ultrathin resin sections
This protocol is taken from the 1999 FEBS course manual and was contributed by Heinz Schwarz.
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<b><font>General remarks:<br />
</font> </b> <font>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. </font>
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<font><b>Note:</b><br />
air-dried resin sections can be stored for months prior to labelling.</font></p>
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<b><font>Procedure:</font> </b><br />
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<font>Encircle sections with a water repellent silicon pen (e.g. PAP-Pen from Electron Microscopy Sciences, Ft. Washington, PA or Sciences Services, Munich). </font></li>
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<font>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. </font></li>
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<font>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. </font></li>
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<font>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. </font></li>
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<font>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. </font></li>
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<font>Use oil immersion objectives to examine.<br />
<b>CAVEAT: </b> All solutions should be centrifuged before use for 2 min. at 10,000 rpm. Avoid air-drying during all incubation steps.</font></li>
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<b><font>References:</font> </b></h3>
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<font><i><font>Immunofluorescence on ultrathin resin sections:</font> </i> </font></p>
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<font>Albrecht U, Seulberger H, Schwarz H, and Risau W. (1990) Brain Res.535, 49-61. </font></li>
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<font>Schwarz H, Hohenberg H, and Humbel BM. (1993) Immuno-Gold Electron Microscopy in Virus Diagnosis and Research (CRC Press, Boca Raton), pp 349-376. </font></li>
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<font>Schwarz H, Müller-Schmid A, and Hoffmann W. (1993) Cell Tissue Res. 273, 417-425. </font></li>
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<font>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. </font></li>
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<font>Fialka I, Schwarz H, Reichmann E, Busslinger M, and Beug H. (1996) J. Cell Biol. 132, 1115-1132. </font></li>
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<font>Kurth T, Schwarz H, Schneider S, and Hausen P. (1996) Cell Tissue Res. 286, 1-12.</font></li>
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<font><i><font>Semi-permanent mounting medium containing polyvinylalcohols (Elvanol/Moviol):</font> </i> </font></p>
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<font>Rodriguez J, and Deinhardt F. (1960) Virology 12, 316. </font></li>
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<font>Lennette OA. (1978) Am. J. Clin. Path. 69, 647-648.</font></li>
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<font><i><font>Antifading agents:</font> </i> </font></p>
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<font><b>1,4-Diazobicyclooctane (DABCO):</b> </font></dt>
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<font>Johnson GD et al, 1982. J. Immunol. Meth. 55, 231-242.<br />
Langanger G, De Mey J, and Adam H. (1983) Mikroskopie 40, 237-241. </font></dd>
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<font><b>Paraphenylendiamine:</b> </font></dt>
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<font>Johnson GD, and Araujo GMDCN. (1981) J. Immunol. Meth. 43, 349-350. </font></dd>
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<font><b>n-Propyl gallate:</b> </font></dt>
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<font>Giloh H, and Sedat JW. (1982) Science 217, 1252-1255.</font></dd>
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