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| A shared resource available for researchers with appointments in the Vanderbilt University Medical Center, supported by the Vanderbilt-Ingram Cancer Center, the Diabetes Research and Training Center, the Digestive Disease Research Center, the Kennedy Center, the Vision Research Center, and the Mouse Metabolic Phenotyping Center. |
This is an example of a protocol which has worked for someone. It may work for you, but be sure to vary what's appropriate for your sample (e.g., the postfixation may not be necessary, but you may need to incubate longer, or with a higher concentration of antibody.) Indirect Immunofluorescence Procedure for Confocal Laser Scanning MicroscopyDr. Gunther Giese, Max-Planck-Institut fur Zellbiologie, Rosenhof, D-68526 Ladenburg, Germany Cells (3x105/slide) are allowed to adhere for 15 min onto polylysine- coated glass coverslips. After a brief rinse with PEM buffer (100 mM PIPES, pH 6.8, 5 mM EGTA, 2mM MgCl2), fixation and permeabilization are carried out in 3.7 % paraformaldehyde, 0.02 % glutaraldehyde, 0.2 % Triton X-100 in the same buffer for 10 min. Following two washes with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM NaH2PO4, 1.8 mM K2HPO4, pH 7.4) free aldehyde groups should be reduced with 0.1 % (w/v)sodium borohydride in PBS, pH 8.0. Non-specific protein binding sites are blocked after two washes with PBS, pH7.4, with 0.5 % (v/v) fetal calf serum and 0.5 % bovine serum albumin in PBS (FCS/BSA/PBS). Incubation with the primary antibody or an antibody mixture is for 1 h, unbound antibody has to be removed by two washes with FCS/BSA/PBS. Secondary antibodies are applied for another hour. After three washes with PBS, postfiixation is done with 3.7 % formaldehyde in PBS, pH 7.4. Samples are rinsed with PBS before and after a treatment with 50 mM NH4Cl in PBS. Before mounting, samples are transferred into PBS, pH 8.9. Mounting media is a 1:1 mixture of glycerol and Vectashield (Vector, Burlingame, CA, USA) containing 2% (w/v) 1,4-diazabicyclo(2,2,2)octan (DABCO, Merck, Darmstadt, Germany). References: Giese et al. 1997. TPA induces apoptosis in MPC-11 mouse plasmacytoma cells grown in serum-free medium. Biology of the Cell 89, 29-41 Bacallao et al. 1990. Guiding principles of specimen preservation for confocal fluorescence microscopy. In: Handbook of Biological Confocal Microscopy (J.B. Pawley, ed), Plenum Press, New York, 197-205 |
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