• International Journal of Oral Biology
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• v.40 no.4
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• pp.183-187
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• 2015

The present study was aimed to evaluate the influence of glutaraldehyde (GA) concentration on multiple electron microscopic (EM) immunostaining using pre-embedding peroxidase and post-embedding immunogold method. Influence of various concentrations of GA included in the fixative on immuoreactivity was assessed in the multiple immunostaining using antisera against anti-transient receptor potential vanilloid 1 (TRPV1) for peroxidase staining and anti-GABA for immunogold labeling in the rat trigeminal caudal nucleus. Anti-TRPV1 antiserum had specificity in pre-embedding peroxidase staining when tissues were fixed with fixative containing paraformaldehyde (PFA) alone. Immunoreactivity for TRPV1 was specific in tissues fixed with fixative containing 0.5% GA at both perfusion and postfixation steps, though the immunoreactivity was weaker than in tissues fixed with fixative containing PFA alone. Tissues fixed with fixative containing 0.5% GA at the perfusion and postfixation steps showed specific immunogold staining for GABA. The results of the present study indicate that GA concentration is critical for immunoreactivity to antigens such as TRPV1 and GABA. This study also suggests that the appropriate GA concentration is 0.5% for multiple immunostaining with peroxidase labeling for TRPV1 and immunogold labeling for GABA.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.4
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• pp.298-304
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• 2006

In this study, we establish a thin-layer chromatography (TLC) immunostaining method for detecting starfish gangliosides. A new monoclonal antibody (MAb) against AG-2, the major gangliosides molecular species of Acanthaster planci, was produced by fusing hybridoma with splenocytes immunized to liposomal AG-2. BALB/c male mice were injected with liposomal AG-2 antigen, and immunized. Their splenocytos were isolated and fused with hypoxanthine-aminopterine-thimidine (HAT)-sensitive mouse myeloma cells. Hybridomas producing MAb reactive to AG-2 were cloned using the limited dilution method. Established hybridomas were cultured in eRDF medium. Crude MAb produced from clone 8D4 was purified with a magnesium pyrophosphate column. Enzyme immunoassay and TLC immunostaining of AG-2 were performed using the purified MAb. Structurally related gangliosides did not cross-react with anti-AG-2 antibodies. The detection limit of TLC immunostaining was 50 ng of AG-2. The newly established immunostaining method was further developed for detecting AG-2 distribution and qualitative analysis in tissues and/or organs. Our results show that the majority of AG-2 is present in the stomach of male A. planci, while AG-2 is distributed not only in the stomach but also in the the pyloric caeca of female A. planci.

• Applied Microscopy
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• v.32 no.3
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• pp.247-255
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• 2002

In this study, we carried out immunostaining and immunogold labeling with antibodies to serotonin and somatostatin to examine the characteristics and functions of the neurons that secrete neurotransmitters in optic lobes of Todarodes pacificus and Octopus minor. As a result of immunostaining with anti-somatostatin, the nerve cells of Todarodes pacificus reacted as similar to the anti-serotonin, but in Octopus minor, only large cells in the outer granule cell layer reacted positively. In the immunogold labeling with anti-serotonin, the nerve cells in the inner grande cell layer and medulla of Todarodes pacificus reacted strongly, 30 gold particles being labeled per $0.5{\mu}m^2$ of the cytoplasm. However, in Octopus minor, only 17 gold particles were labeled, which stated a weak reaction. On the other hand, in the anti-somatostatin case, the nerve cells in the outer and inner granule cell layers and medulla of Todarodes pacificus showed strong reaction, 30 gold particles being labeled per $0.5{\mu}m^2$ of the cytoplasm while the nerve cells in the outer granule cell layer of Octopus minor reacted weakly, about 3 gold particles being labeled per the equivalent area. As a result of immunostaining and immunogold labeling with two types of antibodies to each part of the optic lobes, we found that the reactive nerve cells were distributed differently in the two species. In particular, the degree of reactivity to the immunostaining and immunogold labeling appeared stronger in Todarodes pacificus than in Octopus minor.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6083-6086
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• 2012

Background: The aim of this study was to screen for human papillomavirus (HPV) infections in head and neck squamous cell carcinomas (HNSCCs) using P16 immunostaining. Materials and Methods: A retrospective study was performed on 150 samples from patients diagnosed with HNSCCs. HPV status was determined using $p16^{INK4A}$. Results: 31 of the 150 (20.7%) HNSCCs were HPV positive. Conclusions: A large proportion of HNSCCs in Sudan are associated with HPV infection. The fact that the prevalence of HPV is high among Sudanese patients with head and neck cancers (HNC) has obvious implications for vaccine therapy.

• Proceedings of the KACD Conference
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• 2001.11a
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• pp.561.1-561
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• 2001

The aim of this study was to examine the localization of bacteria in the apices of roots with refractory apical periodontitis by a scanning electron micrscopy(SEM), and to identify the extra-radical bacteria using a immunohistological method. Eleven teeth were prepare for immunostaining and 4 for SEM. Immunostaining was performed with LSAB method and specific antisera against 18 bacteria selected for this study. Specimens for SEM observations were dried using a t-butyl alcohol freeze dryer and coated with gold-palladium.(omitted)

• The Korean Journal of Cytopathology
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• v.9 no.1
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• pp.15-20
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• 1998

Mutant form of the p53 gene product is abnormally accumulated in the nuclei of the tumor cells due to prolonged half life, and readily detected by immunohistochemical methods. To determine the positivity rate of p53 in body cavity fluid according the primary site and histological types of tumors and the utility of p53 immunostaining as an adjunct in the diagnosis of malignancy, we reviewed 69 effusions, including pleural effusion, ascitic fluid, and pericardial fluid, that were diagnosed as overt malignancy and 21 effusions of suspicious malignancy, immunohistochemistry was performed on paraffin-embedded cell blocks using a monoclonal antibody to p53 supressor gene product(Clone DO7) and a standard avidin-biotin complex technique with a citrate buffer antigen retrieval solution. The results were as follows; of the 46 pleural effusions with overt malignancy, 22 were immunopositive for p53 protein; of the 21 ascitic fluids with overt malignancy, 5 were positive for p53. Positivity rates according to the primary sites of tumors were 18 of 34(52.9%), 8 of 21(38.1%), 1 of 9(11.1%) cases of the tumors of the lung, GI tract, and ovary, respectively. According to the histologic types of lung cancer, 11 cases(61.6%) were positive out of 18 adenocarcinomas, 2 of 5 large cell undifferentiated carcinomas, and 1 of 2 small cell undifferentiated carcinomas. Of 21 cases of suspicious malignancy, 6 were positive for p53 and all of them(6/6) were confirmed as adenocarcinoma of the lung or GI tract. These findings indicate that p53 immunostaining using paraffin embedded cell block is useful diagnostic and prognostic marker in body fluid cytology although negative immunostaining does not exclude malignancy.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.1
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• pp.195-200
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• 2013

Secreted protein acidic and rich in cysteines-like protein 1 (SPARCL1), an extracellular matrix glycoprotein, has been implicated in the pathogenesis of several disorders including cancer. However, little is known about the expression and significance of SPARCL1 in human breast cancer. The aim of this study was to determine the expression pattern and clinicopathological significance of SPARCL1 in a Chinese breast cancer cohort. mRNA and protein expression of SPARCL1 in human breast cancer cell lines and breast cancer tissues was detected using the reverse transcription-polymerase chain reaction, real-time quantitative PCR, and Western blotting, respectively. Immunostaining of SPARCL1 in 282 Chinese breast cancer samples was examined and associations with clinicopathological parameters were analyzed. Compared to the positive expression in immortalized human breast epithelial cells, SPARCL1 was nearly absent in human breast cancer cell lines. Similarly, a significantly reduced expression of SPARCL1 was observed in human breast cancer tissues compared to that in normal breast epithelial tissues, for both mRNA and protein levels (P < 0.001). Immunohistochemical analysis showed that strong cytoplasmic immunostaining of SPARCL1 was observed in almost all normal breast samples (43/45) while moderate and strong immunostaining of SPARCL1 was only detected in 191 of 282 (67.7%) breast cancer cases. Moreover, down-regulation of SPARCL1 was significantly correlated with lymphatic metastasis (P = 0.020) and poor grade (P = 0.044). In conclusion, SPARCL1 may be involved in the breast tumorigenesis and serve as a promising target for therapy of breast cancer.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.287-292
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• 2001

Selection of oocyte cryopreseivation method is a prerequisite factor for developing an effective bank system. To develop an effective vitrification method, we examined whether damages in spindle and chromosome morphology induced by vitrification. Intact cumulus-enclosed oocytes were vitrified with DPBS with 5.5 M ethylene glycol and 1.0 M sucrose, and loaded onto eletron microscopic copper grid for storing in liquid nitrogen. Intact vitrified and thawed oocytes were immunostaining for tubulin and karyotying for chromosome. Vitrfied and thawed oocytes had a higher rate of chromosome (32.8% vs. 19.6%) and spindle (32.3% vs. 20.2%) abnormalities compared with fresh oocytes. Mouse oocytes after vitrification at the mature stage showed increased incidence of chromosomal and spindle abnormalities.

• The Korean Journal of Cytopathology
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• v.19 no.2
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• pp.160-163
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• 2008

The cytologic diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) has become one of the common causes of false negative diagnoses when performing fine needle aspiration cytology (FNAC) of the thyroid gland. We retrospectively reviewed all the aspirates for which a diagnosis of FVPTC had been made based on the surgically excised specimens, regardless of the cytologic diagnosis. 145 FNACs was performed in 135 patients. The cytologic diagnoses were categorized as 2 unsatisfactory specimens (1.4%), 16 benign (11.0%), 49 atypical (33.8%) and 78 malignant lesions (53.8%). The tumor cells consistently showed significant nuclear overlapping, irregular nuclei and fine chromatin in all cases; however, nuclear grooves and inclusions were scarce. Galectin-3 immunostaining was performed on the cell blocks of 65 cases and this was positive for 45 cases (69.2%). The results of our study demonstrate that the determination of minimal cytologic criteria is needed to raise the sensitivity of detecting FVPTC by FNAC, and galectin-3 immunostaining is useful to make decisions on the surgical treatment of cytologically atypical thyroid nodules.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.24 no.3
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• pp.295-305
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• 2021

Purpose: Hepcidin levels have previously been reported to be correlated with liver damage. However, the association between hepcidin levels and liver fibrosis in children with fatty liver disease remains unclear. This study therefore aimed to investigate the pathophysiology of fibrosis in children with fatty liver disease and its association with hepcidin levels. Methods: This retrospective case series included 12 boys aged 6-17 years who were diagnosed with nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH) at the Tokyo Medical University Hospital. Sixteen liver biopsy samples from 12 subjects were analyzed. Serum hepcidin levels were assayed using enzyme-linked immunosorbent assay. Immunostaining for hepcidin was performed, and the samples were stratified by staining intensity. Results: Serum hepcidin levels were higher in pediatric NAFLD/NASH patients than in controls. Conversely, a significant inverse correlation was observed between hepcidin immunostaining and Brunt grade scores and between hepcidin scores and gamma-glutamyltranspeptidase, hyaluronic acid, and leukocyte levels. We observed inverse correlations with a high correlation coefficient of >0.4 between hepcidin immunostaining and aspartate aminotransferase, alanine aminotransferase, total bile acid, and platelet count. Conclusion: There was a significant inverse correlation between hepcidin immunoreactivity and fibrosis in pediatric NAFLD patients; however, serum hepcidin levels were significantly higher, suggesting that these patients experienced a reduction in the hepcidin-producing ability of the liver in response to iron levels, leading to subsequent fibrosis. Therefore, hepcidin levels can be used as markers to identify the progression of fibrosis in patients with NAFLD.