Enhanced Polymer One-Step Staining Procedure

Sections (7 xm thick) of freshly frozen tissues are mounted on silane-coated slides and fixed with 4% buffered formaldehyde (pH 7.0) for 20 sec (Richter et al., 1999). The sections are rinsed in TBS (pH 7.4) for 15 sec, followed by incubation with EPOS antibody for 3 min at 37°C in an incubation chamber. They are rinsed twice for 15 sec each in TBS, and then developed with peroxidase-DAB detection kit (Dako) in a microwave oven (500 W) for 1 min during microwaving, the slides are cooled by a cold water bath (Werner et al., 1991). After being rinsed in tap water, the sections are counterstained with hematoxylin for lOsec. They are rinsed in tap water and cover-slipped. 

Tissue specimens are snap-frozen in liquid nitrogen for 30 sec immediately after removal and then transferred to a cryostat (Kammerer et al., 2001). Serial frozen sections of 5 pan thickness are cut and placed on silane-coated slides. They are air-dried for 30 sec, fixed in acetone for 1 min at room temperature (22°C), and air-dried at 22°C (Fig. 6.11). The sections are incubated with primary antibody in the antibody diluent (Dako) for 3 min by placing the slide horizontally on a hot plate at 37°C. (All incubation steps are carried out by placing the slide horizontally on the hot plate at 37°C.) Following a brief rinse in TBS, the sections are incubated with the goat-anti-mouse EnVision-HRP-enzyme conjugate for 3min at 37°C. 

FIGURE 6.11. Step-by-step schematic diagram of the rapid EnVision procedure. RT = room temperature TBS = Tris-buffered saline (pH 7.4) H = Meyer s hematoxylin. Reproduced, with permission, from Kammerer et al. (2001). Copyright 2001 Histochemical Society. 

The sections are rinsed in TBS, and then exposed to DAB+ chromogen (Dako) for a few minutes as the substrate for the EnVision-HRP-enzyme. The sections are washed by shaking the slide rapidly under tap water for 10 sec. The excess fluid is removed from the slide with a paper towel. The slide is dipped in distilled water, counterstained with Meyer s hematoxylin for 15 sec, and then rinsed in hot tap water at 42°C for 30 sec. 

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