Fluorescent counterstaining

Sripanidkulchai, K. Wyss, J. M. Two rapid methods of counterstaining fluorescent dye tracer containing sections without reducing the fluorescence. Brain Res. 1986,397,117-129. 

Counterstaining counterstain nuclei if necessary, e.g., with DAPI for fluorescence microscopy or with hematoxylin (see Sect. 7.4) for brightfield microscopy. 

Fig. 5.2 Immunofluorescent demonstration of smooth muscle actin (FITC, green channel) in the blood vessel wall of the human kidney. Red autofluorescence of erythrocytes, elastic lamellae and kidney tubules was captured with a filter exciting the autofluorescence in red spectrum under a longer exposure than with the filter exciting specific fluorescence in the green spectrum. Nuclei are counterstained with DAPI (blue channel)...

Table 10.1 Characteristics of some fluorescent dyes used for nuclear counterstaining...

Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations (http //probes.invitrogen.com/handbook/sections/0806.html). For instance, Vybrant Apoptosis Assay Kit 4 (Molecular Probes) detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide. 

Labeling with fluorescent phalloidins may be combined with immunostaining. In this case, the phalloidin-staining solution can be applied in a mixture with fluores-cently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell. 

Fig. 11.2 Localization of GFP and calsequestrin (CSQ) in neonatal rat cardiac myocytes. Myo cytes were infected with a recombinant adenovirus containing the cDNAs of GFP and CSQ. Both cDNAs were expressed under control of a separate cytomegalovirus promoter. Expression of CSQ and the coexpressed GFP was detected by fluorescence microscopy. Left-. GFP fluorescence (green), middle immunostaining of CSQ (red), right overlay of GFP fluorescence and immunos taining. Nuclei were counterstained with DAPI (blue). Courtesy of Ulrich Gergs...

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

Fig. 13.3. This figure demonstrates the distribution of fluorescently tagged LCM in brain parenchyma analyzed by confocal laser scanning microscopy. Rats bearing 9L tumors were administered diO-LCM and sacrificed 2 minutes later. Vibratome sections were counterstained with TR-WGA which binds to tumor cells and distinguishes the tumor area from the surrounding normal tissue. Comparison of the area stained with TR-WGA (tumor cells) (B) and that stained with diO (A) indicates that LCM were associated with a large portion of the tumor. (Taken from ref. 531.)...

Immunohistochemical stains use antibodies to identify specific constituents in tissue sections. In order to detect the site of reaction, the antibody is labeled with an enzyme that can be reacted with a suitable substrate to give a colored product. ITie alternative is to use a fluorescent label. The advantage of an enzyme label is that the nuclei can be counterstain ed, thereby revealing the tissue architecture, and that the stain fades slowly, if at all, with time, allowing the slides to be stored. 

Counterstaining in these circumstances also elirmnates the problem that unlabeled regions of the specimen are not normally visible under epifluo-rescence. Without counterstaining, localization of the antigen requires superposition or comparison of a fluorescent image with a phase or... 

To allow visualization of the location of the internalized complexes in relation to the cell nuclei, add 1 pi per well of the cell-permeable nuclear counterstain Hoechst 33342 (1 mg mh stock solution this results in a final Hoechst concentration of 5-lOpg/ml). Incubate for 15-20 min and observe with 350/461 nm blue fluorescence filters for Hoechst dyes (results are shown in Figs. 6a and b). 

Prepare antibleach medium with fluorescent counterstain 90% glycerol and 2.5% l,4-diazabicyclo[2.2.2]oclane (DABCO antifade. Sigma) in IX PBS to which either 1 mg/ml 4,6-diamidino-2-phenylimdole dihydrochloride (DAPI, Sigma) or 5 mg/ml propidium iodide is added. 

In some samples, the occurrence of fluorescent particles may lead to an aborted scan or to a cumbersome validation when numerous spots are present in the secondary scan map. By adding a counterstaining step (Catala et al. 1999) and/or using a filter with a larger pore size, this problem can sometimes be overcome. Additionally, the fluorescence intensity of cells labelled using FISH or immunofluorescence is often low, making signal amphfication necessary (see Section Fluorescent Stains for SPC). 

Sulfur mustard adducts were clearly detected in the stratum corneum whereas DNA counterstaining visualizes the presence of DNA in the nucleated cells. Hardly any fluorescence due to antibody treatment was measured over the nonexposed skin cross-section at the conditions used. In contrast to the immunochemical method for analysis of DNA-sulfiir mustard adducts, which involves laborious workup procedures, this approach opens the way for development of a rapid detection kit that can be applied directly to the skin. 

During the FISH procedure, DNA within cells placed on a slide and the fluorescently labeled probe of interest are denatured by incubation at high temperature. Then the probe is allowed to hybridize to the target DNA. The next step is a series of post-hybridization washes to remove the probe excess. Finally, after counterstaining the nuclei, the probe signal can be visualized under a fluorescent microscope. 

DAPI (4, 6-Diamidine-2-phenylindole dihydrochloride), a fluorescent stain that specifically binds double stranded DNA, is widely used to specifically localize nuclei in developing embryos and as a counterstain. Phalloidin is a plant toxin that binds filamentous actin, thus allowing the visualization of cell borders by highlighting the F-actin network along cell membranes. Both are toxic and should be handled with care. 

Fluorescent nuclear counterstains such as DAPI (blue), or 7-AAD (red) can substantially improve recognition of morphological details. Dilute any of these dyes 1 1000 in TBS and incubate between 30 s and 2 min. 

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