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Staining toxoplasma gondii with fluorescein-labelled antibody. II. A new serologic test for antibodies to Toxoplasma based upon inhibition of specific staining.

A new serologic test for antibodies to Toxoplasma is described, which is based upon inhibition of specific staining with fluorescent antibody. In performing the test, a mixture of the test serum and known fluorescein-labelled antiserum is added to a dried smear of toxoplasms for 1 hour at 37 degrees C. The smear is then rinsed and examined with a fluorescence microscope. Reduction in the brightness of fluorescence, as compared to that of a negative control slide, indicates the presence of antibody in the test serum. A comparison of the results of this test with those of the methylene blue dye test showed a strong parallelism between the two sets of results. On the other hand, the complement-fixation test for toxoplasmosis did not yield nearly as many positives as the inhibition test. The specificity of the new test was studied by comparing it with dye test results and clinical histories in human patients, and by testing a group of animals immunized with a variety of non-Toxoplasma antigens. No evidence of cross-reactions was obtained in the latter series. Some advantages and disadvantages of the inhibition test are discussed.

Animals↗

The histochemical demonstration of protein-bound sulfhydryl groups and disulfide bonds in human hair by a new staining method (DACM staining).

A new fluorogenic maleimide DACM, i.e., N(7-dimethyl amino-4-methyl coumarinyl) maleimide, does not fluoresce by itself. It is specifically combined with --SH groups and becomes fluorescent (lambda ex: 400 nm, lambda em: 485 nm). At this range of excitation the frozen skin section has no native fluorescence because the emission maximum of DACM does not overlap with any of aromatic residues of proteins such as tryptophane. S--S groups can be demonstrated with DACM by inhibiting native --SH and then reducing SS to --SH. --Sh was generally abundant at the bulb region. --SH of hair cortex was more concentrated at keratogenous zone. Further up in the follicle, --SH of hair cortex was gradually decreased, although at the region of isthmus, --SH reaction of the hair cortex was still moderately strong. Outer root sheath had --SH from the upper bulb to the surface of the epidermis. One the other hand, no S--S was demonstrable at the bulb region. At the level of keratogenous zone, however, S--S linkages of hair cortex and inner root sheath began to appear and further up in the follicle S--S linkages were increased gradually. Outer root sheath had no S--S linkages by DACM staining up until it is keratinized along the hair canal in the upper follicle. The concentration of --SH and S--S thus seemed to be reciprocal; --SH is found in non-keratinized tissues, whereas S--S is abundant in keratinized areas. These findings are at some variance with conventional data, which suggest, for example, that --SH groups disappear suddenly above the keratogenous zone.

Adult↗

Immunoperoxidase staining of previously stained tissue.

Using an unlabelled antibody peroxidase-antiperoxidase method, we have demonstrated that certain tissue antigens can be identified in tissue sections previously stained with hematoxylin and eosin. This technique is useful in identifying antigens in tissues where insufficient unstained material is available.

Antigens↗

Pre-embedding staining for GAD67 versus postembedding staining for GABA as markers for central GABAergic terminals.

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for gamma-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.

Animals↗

Detecting p53 gene mutation of breast cancer and defining differences between silver staining PCR-SSCP and immunohistochemical staining.

This study detects and defines the patterns of p53 gene mutations in breast cancers. We analyse p53 gene mutations through comparing the results of single-strand-conformation-polymorphism (SSCP) and immunohistochemistry (IHC), and we try to define the differences between the results of SSCP and IHC. Twenty-seven fresh primary breast cancer tissues and eight normal breast tissues were studied. The IHC was done with the usual streptavidin-biotin peroxidase complement method by using monoclonal antibody DO-7. The results of staining was scored. The SSCP method was done by using Cold SSCP Electrophoresis System. Overexpressions of p53 protein were seven (25.9%) among 27 cancer cases on IHC. Four (57.1%) of seven cases were positive in SSCP. In SSCP, the mutations were detected in 10 (37%) among 27 cancer cases. The mutations were two in exon 5, one in exon 8, and seven cases in exon 7. All of 10 mutations were proved by sequencing analysis. Of them, only four (40%) were positive in IHC. We consider the IHC as a screening method for p53 gene mutations.

Adult↗

[Two cases of ulcerative colitis with colon cancer: immunoperoxidase staining using monoclonal antibodies against gastrointestinal tumor and mucin staining].

Surgical specimens from 2 patients with chronic ulcerative colitis accompanied with colon cancer were evaluated by immunoperoxidative staining using monoclonal antibodies A7 (against human colon cancer), S202 (against human gastric cancer), and anti-carcinoembryonic antigen (CEA). The three monoclonal antibodies were reactive with cancerous tissue, anti-CEA antibody and monoclonal antibody S202 reacted with dysplasia tissues, whereas monoclonal antibody A7 did not. Using high-iron diamine technique for mucosubstances (sialomucin and sulfomucin), cancer and dysplasia showed no secretory elements. Surrounding mucosa showed both sialomucin and sulfomucin secretion.

Adenocarcinoma↗

[Identification of simian chromosomes by the differential staining method with Romanovskii-Giemza stain. II. Papio hamadryas].

By means of differential staining with the Romanovsky - Gimsa dye the distribution of G-bands was studied in chromosomes of bone marrow cells and of cultured peripheral blood lymphocytes in Papio hamadryas. All the homologous chromosome pairs were identified. It was shown, as compared Papio hamadryas karyotypes with that of Macaca mulatta, that patterns of most chromosomes differentiation were similar. Certain differences between these species were observed in the character of differentiation of chromosomes 1 and 5 and also in parameters of chromosomes 2 and 21 (X).

Animals↗

[Identification of monkey chromosomes by differential staining with Romanovsky--Giemza stain. III. Cercopithecus aethiops].

The distribution of the G-bands in chromosomes of bone marrow cells of Cercopithecus aethiops was studied by means of differentail staining with the Romanovsky - Gimsa dye. All the homologous chromosome pairs were identified and morphometric parameters were detected. The comparison of C. aethiops karyotypes with those of Macacca mulatta has shown that they are different in numbers and in the character of banding pattern of the most chromosomes. Both species revealed 12 pairs of chromosomes similar in their morphology and parameters.

Animals↗

[Morphological study of muscle fibers stained red by modified Gomori trichrome staining with special reference to smooth red fibers].

The modified Gomori trichrome stain of muscles can demonstrate ragged red fibers which are irregular in outline and display a thick and irregular red subsarcolemmal layer and intermyofibrillar red deposits. Typical ragged red fibers are often encountered in mitochondrial myopathy. On the other hand, we have noticed fibers outlined by a thin red subsarcolemmal layer. These fibers are smooth in outline. The sarcoplasm shows normal intermyofibrillar network. We defined these fibers as "smooth red fibers". To investigate the pathological significance of the smooth red fibers, we studied morphological differences between the smooth red fibers and ragged red fibers by light and electron microscopy and evaluated the occurrence and characteristics of the both abnormal muscle fibers in several neuromuscular diseases. Muscle specimens from 738 patients who were seen or consulted at the Department of Neurology, Hokkaido University, from January 1980 to October 1994 were examined. The smooth red fibers were classified into two types, type I and type II. Type I smooth red fibers were hypertrophied and showed a thin smooth red margin. Electron microscopy of the type I smooth red fibers showed no mitochondrial abnormality, being different from ragged red fibers which have abnormal mitochondria. Type I smooth red fibers were observed in chronic denervation process; they were specially frequent in Kugelberg-Welander syndrome. Hypertrophy of type I smooth red fibers were considered to be a compensative reaction in chronic denervation. Type II smooth red fibers were observed with or without ragged red fibers in mitochondrial myopathy. Type II smooth red fibers showed a thin smooth red margin, spreading red deposits from the margin into sarcoplasm. The fibers showed mitochondrial abnormality in electron microscopy. It could be posturated that type II smooth red fibers were transformed into ragged red fibers. The findings suggest 1) type I and type II smooth red fibers are different in origin, 2) type II smooth red fibers change to ragged red fibers, 3) type I and type II smooth red fibers are important markers of chronic denervation and mitochondrial myopathy, respectively. These two types of smooth red fibers are worthy of attention in muscle pathological observations.

Adolescent↗

Studies on Papanicolaou staining. I. Visible-light spectra of stained cervical cells.

This paper presents visible-light absorbance spectra of the nuclei and cytoplasms of ten types of cervical cells stained by the Papanicolaou technique, together with tables summarizing the data. It is concluded that for automated image processing of Papanicolaou smears the most suitable wavelengths are 531 nm to maximize the contrast of cell against background and a wavelength in the range 560 nm to 605 nm (with a mean at 575 nm) to maximize the contrast of the nucleus against the cytoplasm.

Carcinoma in Situ↗

Immunohistochemical staining of ras oncogene product in neoplastic and non-neoplastic mesothelial tissues: immunoreactivity for N-ras and lack of immunohistochemical staining for Ha-ras and K-ras.

Immunohistochemical staining of 36 malignant mesotheliomas and 45 cases of non-neoplastic mesothelium including 20 specimens with signs of hyperplasia were investigated using murine monoclonal antibodies directed against p21 ras protein, Ha-ras protein, K-ras protein, and N-ras protein. All cases of non-neoplastic mesothelium and the majority of the malignant mesotheliomas (78 per cent) showed cytoplasmic and often submembranous immunoreactivity in more than 50 per cent of the cells with both the pan-ras and N-ras antibody. No immunoreactivity was observed for Ha- and K-ras. There was no statistically significant difference with respect to immunoreactivity between neoplastic and non-neoplastic mesothelium or between the various mesothelioma subtypes.

Adult↗

[Typing of gonadospecific diaphorase (DIA3) in seminal stains and esterase D typing in seminal and saliva stains (author's transl)].

1. The gonadospecific DIA3 types were traceable for 4 weeks, in some cases for 6 weeks. 2. EsD polymorphism was not demonstrable by starch gel electrophoresis in sperm, not even in sperm lysates. 3. EsA patterns showed only non-reproducible differences. 4. Because of their low concentrations, salivary esterase cannot cause errors when mixed stains are examined.

Electrophoresis, Starch Gel↗