Hoechst mounting

Mounting mount sections in aqueous medium or balsam for brightfield microscopy or in anti-fade medium for fluorescence microscopy (see Sect. 3.2.2). Notes. A11 incubations are at room temperature unless otherwise noted. Nuclear dyes (DAPI, Hoechst 33342 and Propidium Iodide) supplied as lyophilized solids are usually reconstituted in methanol. The stock solutions (5 mg/ml) are stable for many years when stored frozen at <—20°C and... 

Stain slides with Hoechst 33258 for 10 min, and mount in antifade solution. 

Rinse with PBS and mount the coverslip as described above. This method, using unfixed preparations, is even more rapid than the one described above using Hoechst 33258. A kit based on DAPI is available from Bioassay Systems. 

Note Hoechst 33258- and DAPI-stained slides can also be mounted in Vectashield H-1000. However, as mentioned earlier, Vectashield affects the appearance of cells viewed by phase-contrast optics. 

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. 

Both primary and secondary antibodies are diluted in PBS. After the incubation in secondary antibodies, sections are washed in PBS, incubated with 0.1 pg/mL Hoechst 33258 for 5 min, washed again with PBS, and finally mounted in Mowiol for analysis under conventional fluorescence or laser confocal microscopy. 

For confocal laser scanning microscopy, we use a Leica TCS-SP system mounted on a Leica DMIRBE inverted microscope for fluorescence excitation, an Ar UV laser at 364 nm is used for Hoechst 33258 and Ar visible laser at 488 nm for Alexa-Fluor 488 and He/Ne laser at 543 for Alexa-Fluor 594. Spaced (0.5 pm) optical sections are recorded using a 63x oil immersion objective. Images are collected in the 1,024 x 1,024 pixels format, stored on a magnetic mass memory, and processed by the Leica Confocal Software. 

Fig. 9.1. Surface-mount miniature connector made from Vectra (by courtesy of Hoechst-UK Ltd, Polymers Division).

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