| 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 (0.2gelatin, 0.5BSA 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: |
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