Double immunofluorescence staining

Basic protocol for double immunofluorescence staining using primary antibodies raised in two different host species... 

Shindler, K. S., and Roth, K. A. 1996. Double immunofluorescent staining using two unconjugated primary antisera raised in the same species. J. Histochem. Cytochem. 44 1331-1335. 

Fig. 31 Double-immunofluorescent staining of 20-day rat cerebellar sections with antibodies to GD, gangli-oside and glial acidic fibrillary protein (GFAP). Purkinje cell dendrites are intensely GDj-immunoreactive (A) but do not extend to the pial surface, unlike the Bergmann glial fibers (B), which project brightly GFAP-immunoreactive end-feet onto the pial membrane. Scale bar is 35 /tm. Reynolds and Wilkin (1988).

As stated above, this approach is applicable only when primary antibodies are raised in different species. For double or multiple indirect immunofluorescence staining with primary antibodies raised in the same species, see Sects. 8.2. and 8.3 below. 

Frisoni GB, De Leo D, Rozzini R, et al Psychic correlates of sleep symptoms in the elderly. Int J Geriatr Psychiatry 7 891-898, 1992 Fritschy J, Benke D, Mertens S, et al 5 types of type A GABA receptors identified in neurons by double and triple immunofluorescence staining with subunit specific antibodies. Proc Natl Acad Sci USA 89 6726-6730, 1992 Frye CA, Duncan JE Progesterone metabolites effective at the GABAa receptor complex attenuate pain sensitivity in rats. Brain Res 643 194-203, 1994 Erye CA, Cuevas CA, Crystal S, et al Diet and estrous cycle influence pain sensitivity in rats. Pharmacol Biochem Behav 45 255-260, 1993 Erye PE, Arnold LE Persistent amphetamine-induced compulsive rituals response to pyridoxine (B6). Biol Psychiatry 16 583-587, 1981... 

MICROWAVE HEAT-ASSISTED DOUBLE INDIRECT IMMUNOFLUORESCENCE STAINING... 

Fritschy JM, Benke D, Mertens S, Oertel WH, Bachi T, et al. 1992. Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies. Proc Natl Acad Sci USA 89 6726-6730. 

Mixing of colors is less easily detected with enzyme methods than with double immunofluorescent labeling where filter systems in the microscope are more effective in segregating the staining intensities of the two labels. The use of fluorescent antibodies and enzyme-labeled antibodies for the different epitopes may lend itself particularly well to assess co-distribution of epitopes. 

Fig. 2. Treatment of cells with CT976 inhibits ER export of VSV-G, which accumulates in COPII buds at ERES. Cells were infected with ts045 VSV at 40° to accumulate VSV-G in the ER, subjected to various chase protocols, and then stained by double-immunofluorescence for VSV-G (left panels) and the Golgi marker a-mannosidase II (Man II) (right panes). (A, B) No chase (C, D) Cells shifted to 32° for 15 min (E, F) Cells shifted to 32° in the presence of CT976 (G, H) Cells shifted to 32° in the presence of CT976 for 15 min and then washed free of the drug for an additional 15 min. Arrows in E indicate accumulation of VSV-G in foci corresponding to ERESs.

Figure 12. Immunohistochemistry of enterotoxin binding sites with synaptophysin- positive sites. A phrenic nerve-diaphragm preparation showing inhibition of neuromuscular transmission after the treatment with the enterotoxin was stained by a double immunofluorescent method, a, b, and c show the fluorescence of FITC conjugated anti-enterotoxin antibody, d, e, and f show the fluorescence of rhodamine conjugated anti-synap-tophysin antibody corresponding to the sites of a, b, and c, respectively. Note the coincidence of configurations of anti-enterotoxin fluorescence and those of anti-synaptophysin.

Tomehave, D., Hougaard, D. M., and Larsoon, L.-L. 2000. Microwaving for double indirect immunofluorescence with primary antibodies from the same species and for staining of mouse tissues with mouse monoclonal antibodies. Histochem. Cell Biol. 113 19-23. 

Predictions for the structure of DCrn7 display a double propeller structure as it was shown for the C. elegant Actin-Interacting-Protein-1 (AIP-1)7 Analysis of DCrn7-GFP fusions and immunofluorescence studies with specific monoclonal antibodies show a localization to actin rich structures in the cells and dot like staining in the cytosol. This contrasts with the location described for the mammalian protein which was primarily found on the Golgi apparatus. °... 

Figure 3 Uptake of PLGA nanoparticles by murine DCs in vivo. DCs were isolated from the draining lymph nodes of mice administered with nanoparticles containing TMR dextran. Cells were examined for double color immunofluorescence after staining with the following FITC-labeled antibodies (A) DEC-205, (B) DEC-205 isotype control, (C) CD86, (D) CD86 isotype control. Negative controls consisted of unlabeled cells with (E) and without (F) TMR dex-tran-loaded nanoparticles. (From Ref. 128.)...

Wtirden, S. and Hombetg, U. (1993) A simple method for immunofluorescent double staining with primaiy antisera from the same species. J. Histochem. Cytochem 41, 627-630. 

The fate of other nuclear components can concomitantly be analyzed using double-label immunofluorescence microscopy. Cells were fixed and air dried (see above), then incubated with antiguinea pig-specific secondary antibodies conjugated to FITC (10 min), and afterward washed in PBS (10 min). In a second step, the cover slips were incubated for 20 min with an antibody specific for a different nuclear protein (for example, a mouse monoclonal antibody against nucleolar protein fibrillarin). After washing in PBS (10 min), the cells were incubated with an appropriate secondary antibodies (in this case, antimouse-speciflc antibodies) conjugated to Texas Red (Dianova) for 10 min. For the visualization of chromatin, specimens were stained with the DNA-specific fluo-rochrome Hoechst 33258 (5 jug/ml in PBS) simultaneously with the secondary antibody. Finally, the cover slips were washed for 10 min in PBS, dehydrated in ethanol, and embedded in Mowiol (Hoechst, Frankfurt, Germany). 

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