Hoechst stain

Plate storage. We found that using aluminum seals (Fisher Scientific, Pittsburgh, PA, cat. no. 072-00684) allowed us to store plates at 4°C. The use of opaque seals enables plates to be imaged in the presence of ambient light. In our experience, the Hoechst stain and GFP signal remains stable at 4°C for up to 3 mo. 

FIGURE 8.32 (a) A bright field epi-fluorescence image of two polymorphonuclear white cells (probably neutrophils), (b) The same cells shown in (a) imaged via the Hoechst stain. Note the differences in conformation between the two nuclei. The cell on the top is stuck at the entrance to a channel, and the cell at the bottom is being deformed into a channel. The channels are coated with polyurethane to reduce cell adhesion and to enhance white cell penetration [1175], Reprinted with permission from Springer Science and Business Media. 

Hoechst stain stock solution (100 ml) bisbenzimide-Hoechst 33258 ... 

Wrap the container in aluminium foil and store in the dark at 4°C Hoechst stain working solution (50 ml)... 

Hoechst stain working solution (prepare fresh as required) Mountant... 

Add 2 ml of Hoechst stain (wearing gloves) to the coverslip and leave for 5 min at room temperature. Shield the coverslip from direct light at this point. 

Removal of broken cells debris before Hoechst staining... 

Hoechst stain stock solution (100 mL) Add 10 mg Bisbenzimide Hoechst 33258 to 100 mL of distilled water and allow to dissolve. Filter sterilize using a 0.2 pm filter unit. Wrap the container in aluminum foil and store in the dark at 4°C. NB The toxic properties of Hoechst 33258 are unknown therefore, gloves should be worn at all times when handling the powder or solutions. 

Hoechst stain working solution (50 mL) Add 50 pL of stock solution to 50 mL of distilled water. Prepare immediately before use. 

Wearing gloves, return the coverslip to the dish and add 2 mL Hoechst stain (working solution). Shield the coverslip from direct light and leave at room temperature for 5 min. 

Test the culture for the presence of mycoplasma by a Hoechst stain. If mycoplasma is still detectable, it is unlikely that this antibiotic will be successful and an alternative should be tried on a fresh batch of cells. 

If the Hoechst stain gives a negative result, then the cells should be cultured in antibiotic-free medium for a period required to conduct 10 passages. Testing should be conducted at every passage to monitor treatment success since mycoplasma may persist at low levels immediately after antibiotic treatment. 

Cover sections with diluted Hoechst stain (1 500 of 1 mg/ml Hoechst 33258 dye in DMSO) and incubate for 2 min. 

Figure 4. Localizarion of US28 (US28-YFP fusion protein) in transiently transfected human foreskin fibroblasts coinfected with theTowne strain of HCMV, 24 hours post infection (A and B) and 48 hours post infection (C and D). Antibody against HCMV lEf (red staining) (D and F) is used to detect HCMV infected cells. Hoechst staining (blue) (C and E) show all nuclei. Scale bars are 30 pm. Transfections, infections and deconvolution images are prepared by TN Kledal as described in ref. 72.

Use of Hoechst stain in the extract is not mandatory but facilitates microscopic observations (Fig. 1 see Section VIII). 

Figure 3.1 Comparison of HeLa cells stained with FCDs (a) and Hoechst stain (b).

HeLa cells stained with Hoechst stain show void regions (marked by red circles). Scale bar = 10 pm. Reproduced from ref. 100b with permission from the Royal Society of Chemistry. 

Drain the slides, add a solution of Hoechst stain, and apply a large coverslip. 

Fig. 24 An indium bis(thiosemicarbazonato) complex (b) observed in HeLa cells using con-focal microscopy (a) by Pascu et al, indicating nuclear uptake by Hoechst staining (blue), where green signifies the complex and cyan denotes colocalisation. ...

Solution of appiopiiate second antibexly conjugated to a suitable fluorochrome diluted according to the manufacturer s recommendations in FBSIB 10 p.M Hoechst stain (Hoedist No. 33258) in PBS this is conveniently prepared from a stock solution of the dye (1 mM in water, stored at -80 C)... 

The following procedure (Gatti et al. 1976 Gatti and Pimpinelli 1983) is a modification of a protocol developed by Latt (1973) for mammalian chromosomes. For other Hoechst-staining protocols, see Holmquist (1975) and Hazelrigg et al. (1982). 

Notes The Hoechst stain is for quality control of the DNA denaturation step. The euchromatin of interphase chromosomes produces a fuzzy Hoechst stain if properly denatured. Heterochromatin and mitotic chromosomes stain fairly well, despite the denaturation. 

Stain slides in Hoechst staining solution for 5 minutes. 

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. 

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