Hoechst fluorescence counterstaining

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). 

Plate 21 A fluorescent molecular probe. Fixed and permeabilized osteosarcoma cells were simultaneously stained with the fluorescent lectins, Alexa 488 concanavalin A (Con A) and Alexa 594 wheat germ agglutinin (WGA). Con A selectively binds a-mannopyranosyl and a-glucopyranosyl residues, whereas WGA selectively binds sialic acid counterstained with blue-fluorescent Hoechst 33342 nucleic acid stain. See Fluorescent Molecular Probes, Inc. 

Chromosomes with fluorescent signals can be counterstained with Hoechst 33258 as described below. However, banding patterns produced by the fluorescent DNA stains are often difficult to compare with the published polytene chromosome-banding patterns. We find it more convenient to simply use phase contrast to correlate the fluorescent signal with the corresponding chromosome bands. 

Denaturation of the DNA The main drawback of the denaturation of the DNA is that the harshness of the treatment can limit the ability to detect phenotypic markers of interest. Experimenters have developed all sorts of alternatives to deal with this particular problem. Among these is precisely the new non-halogenated thymidine analog, EdU. Salic and Mitchison [22] describe the development of EdU on sections of mouse brain already mounted on glass slides. After the removal of paraffin, sections were stained with 10 pM Alexa 568 azide for 10-30 min, after previous incubation for 10-30 min with 100 mM Tris + 0.5-1 mM CUSO4 and 50-100 mM ascorbic acid (added last). In their case, they counterstained the tissue with Hoechst stain, yet another of the bis-benzimide blue fluorescent dyes used to stain DNA. 

Fig. 7 IMF of activated caspases. Immunocytochemical reaction for activated caspase-9 (a), caspase-8 (b), and caspase-3 (c) in cisPt-treated neuroblastoma (B50) cells (green fluorescence). DNA is counterstained with Hoechst 33258 (blue). Scale bars 20 pm...

Fig. 8 Confocal fluorescence microscopy of B50 neuroblastoma cells, (a, b) Immunolabeling of mtHSP70 green) in B50 cells in controls (a) and cisPt-treated cells (b). After cisPt, mitochondria are clustered around the nucleus and form dense masses in the cytoplasm (b). Nuclei are counterstained with Hoechst blue), (c, d) Double immunolabeling of filamentous actin red) and a-tubulin green) in B50 control cells (c) and in 48 h cisPt-treated cells (d). CisPt-induced cytoskeleton damage leads tubulin to reorganize into thick bundles (e) and to disruption of filamentous actin microfilaments and accumulation of depolymerized actin at cell periphery (f).

Fig. 10 Confocal microscopy double IMF of neuroblastoma B50 cells. Staining for mitochondria green) and lysosomes (red) in (a) controls, (b) after 48-h treatment with cisPt, and (c) recovery. Images show that, after cisPt, lysosomes and mitochondria are distributed in cytoplasmic clusters compared to control, but do not show colocalization. During recovery, cells assume a morphology similar to control cells and show an increase of lysosomes. Immunohistochemistry for LC3B (green) and lysosomes (red) in (d) control and (e) recovery cells. Graph analysis shows the fluorescence peak and colocalization of the two labels. DNA is counterstained with Hoechst 33258 (blue). Scale bars 20 pm...

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