Nuclear stain

Kem-eisen, n. mottled white pig iron (Elec.) core iron, -eiweisskorper, -eiweisstoff, m. nucleoprotein. -farbe,/., -f bemlttel, n., -farbstoff, m. nuclear stain, -f bung, /, Micros.) nuclear staining, -faser, /. nuclear fiber or filament. 

Another morphological assay of apoptosis is done with acridine orange, a nuclear staining that reveals chromatin condensation under light and fluorescent microscope. 

Figure 1.1 Examples of immunostaining for c-Fos IR (dark nuclear staining) and GAD (light cytoplasmic staining) in the MnPN. A higher percentage of GAD-positive cells (arrows) also expressed c-Fos IR (black arrows) following high (A) compared with low spontaneous sleep (B). From Gong et al (2004b). See also Plate 1.

Figure 1.2 Examples of immunostaining intensity from comparison of pRB-IHC in 27 cases of FFPE tissues of bladder cancer (Table 1.4). (A-D) Negative (<10%) showing a few weak positive nuclei (arrows) (E-H) moderate positive (>10%) (I-P) strong positive (>50%). Arrows indicate positive nuclear staining for some lymphocytes or other stromal cells as an internal control. Note the lack of nuclear hematoxylin counterstaining due to low pH AR treatment. The order of cases are indicated in Table 1.4. Reproduced with permission from Shi et al.. Biotech. Histochem. 2007 82 301-309. See color insert.

Weak false-positive nuclear staining may occur with some monoclonal or polyclonal antibodies therefore, careful assessment of the reaction pattern with particular attention to the staining of the normal cells is recommended. False-positive nuclear staining may be accentuated if the slides are allowed to dry during the staining procedure. 

If propidium iodide is used as the nuclear stain, then the incubation time at 4°C overnight yields the best results. Although the cell suspension may be examined after 30-45 min, the longer the propidium iodide is allowed to react with the DNA, the better the staining. 

DAB- Cover the section with this substrate, and incubate the slide for 5 min The product is brown, and is stable m alcohols and in xylene. Counterstain with Mayer s hemalum for 5 mm Wash in tap water. Dip the slide in saturated lithium carbonate for a few seconds this makes the nuclear stain blue. Wash m tap water Dehydrate through ethanol and xylene (or Histoclear), and mount in a permanent mountant, e.g, DPX... 

Colakovski H, Lorber DL. Propylthiouracil-induced peri-nuclear-staining antineutrophil cytoplasmic autoantibodypositive vasculitis in conjunction with pericarditis. Endocr Pract 2001 7(l) 37-9. 

The DNA in the sections is denatured by treatment with 70% formamide/2 x SCC for 5 min at 80°C. Ten microliters of the probe solution (hybridization buffer 7 pd, probe 1 pi, and distilled water 2 pi) is placed on the slide and coverslipped. The slide is placed in a microwave oven (2.45 GHz, 300 W) and heated for 3 sec at 2-sec intervals for a total of 15 min at 42°C. DAPI II (4,6-diamidine-2-phenylindol) (125 ng/ml) is used for nuclear staining. The sections are promptly observed under a fluorescent microscope equipped with epifluorescence filters and a photometric CCD camera. The captured images are digitized and stored in an image analysis program. 

Dried mature green conidia were added to 50 mL of the medium for swelling in a 100-mL Erlenmeyer flask and incubated for 6 h at 28°C using a rotary shaker (TAITEC R-30 mini Taitec, Koshigaya, Japan) (160 rpm). The changes in conidium and nucleus were then observed using a microscope (BH-2, BX-50 Olympus) with or without nuclear staining. 

Swollen conidia were added to 25 mL of the medium for autopolyploidization in a 50-mL Erlenmeyer flask and incubated statically for 7 d at 28°C. The nuclear changes were then observed by nuclear staining using microscopes. 

Whether unoccupied GR are totally nuclear is less clear. Immunocytochemical studies with anti-GR antibodies show both cytoplasmic and nuclear localization at the light level, and an increase in nuclear staining simultaneous with decreased cytoplasmic staining in response to addition of glucocorticoids [106-109]. It may well be that GR have a different intracellular distribution from the sex-steroid receptors. 

Fig. 1. Examples of positive immunostaining. (A) Cytoplasmic staining of rat Purkinje cells by human serum diluted 1 1000, from an individual with paraneoplastic cerebellar degeneration and Yo antibodies. (B) Serum from an individual with paraneoplastic encephalomyelitis and Hu antibodies, diluted 1 500, showing nuclear staining of rat Purkinje cells. (C) Serum from the same individual as in (B), staining the neuronal nuclei of dorsal ganglion from rat, with nucleolar sparing.

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