Observation of dendrite osteocytes of mice at different developmental stages using Ploton silver staining and phalloidin staining / 南方医科大学学报

OBJECTIVE@#To assess the value of Ploton silver staining and phalloidin-iFlour 488 staining in observation of the morphology of osteocyte dendrites of mice at different developmental stages.@*METHODS@#The humerus and femurs were harvested from mice at 0 (P0), 5 (P5), 15 (P15), 21 (P21), 28 (P28), and 35 days (P35) after birth to prepare cryo-sections and paraffin sections. HE staining of P35 mouse femur sections served as a reference for observing osteocytes in the trabecular bone and cortical bone. The humeral sections at different developmental stages were stained with Ploton silver staining to observe the morphology of osteocytes and canaliculi, and the canalicular lengths in the cortical and trabecular bones of the humerus of the mice in each developmental stage were recorded. The cryo-sections of the humerus from P10 and P15 mice were stained with phalloidin iFlour-488 to observe the morphology of osteocytes and measurement of the length of osteocyte dendrites in the cortical bone.@*RESULTS@#In the trabecular bone of the humerus of P0-P15 mice, Ploton silver staining only visualized the outline of the osteocytes, and the morphology of the canaliculi was poorly defined. In P21 or older mice, Ploton silver staining revealed the morphology of the trabecular bone osteocytes and the canaliculi, which were neatly arranged and whose lengths increased significantly with age (P21 @*CONCLUSIONS@#Mouse osteocyte dendrites elongate progressively and their arrangement gradually becomes regular with age. Ploton silver staining can clearly visualize the morphology of the osteocytes and the canaliculi in adult mice but not in mice in early stages of development. Phalloidin iFlour-488 staining for labeling the cytoskeleton can be applied for mouse osteocytes at all developmental stages and allows morphological observation of mouse osteocytes in early developmental stages.

Observation on the distribution of nerve fibers and neural cells morphology in Aspidogaster conchiola / 中国血吸虫病防治杂志

Objective To understand the distribution of nerve fibers and the types of neural cells in Aspidogaster conchiola. Methods Whole worms were subjected to silver staining, histochemical staining and hematoxylin-eosin (HE) staining, and the nervous systems of the worms were observed. Results There were 3 types of neural cells in the worm head near the cerebral ganglion, including unipolar, bipolar and multipolar neurons, which were divided into 7 types according to the morphology. There was a nerve network on the surface of pharynx and intestinal tract, as well as the reproductive organ, including testis, ovary, lower uterus and penis sac. The nerve network was consisted of circular and longitudinal nerve fibers, and the structure of the nerve network around the mouth was similar to central nerve. Conclusions The structure of the A. conchiola central nervous system is very complicated, and the neural networks may be associated with the physiologic activity of the worm. Different neural cells may have diverse functions.

Genotyping of Treponema pallidum in patients with early syphilis in Guangxi province / 中华皮肤科杂志

Objective To investigate genotyps of Treponema pallidum (Tp) in several cities in Guangxi province. Methods A total of 300 patients with suspected early syphilis were enrolled from STD clinics in Guangxi between January 2012 and July 2016, and tissue fluid samples were collected from skin lesions. Silver staining was performed to detect Tp, and PCR to amplify the Tp polA gene for the diagnosis of early syphilis. Positive samples were subjected to PCR amplification of a 60-bp tandem repeat region within the arp gene, restriction fragment length polymorphism(RFLP)analysis of the tpr Ⅱgene after digestion with Mse Ⅰ enzyme and tp0548 genotyping. Results Finally, 215 patients were diagnosed with early syphilis, including 210(97.7%)patients positive for PCR and 105(48.8%)patients positive for silver staining, and the positive rate significantly differed between the two methods (χ2 = 103.01, P < 0.05). Among the PCR-positive samples, 190 could be genotyped by analysis of three target genes, and 17 genotypes were identified. The genotype 14d/f was predominant (45.3%, 86/190), followed by 15d/f (13.7%, 26/190), 16d/f(11.6%, 22/190), 17d/f(7.4%, 14/190), 13d/f(6.8%, 13/190), 10d/f(4.2%, 8/190), 18d/f(1.6%, 3/190), 16a/f(1.6%, 3/190), 5d/f(1.1%, 2/190), 7d/f(1.1%, 2/190), 12d/f(1.1%, 2/190), 16d/e(1.1%, 2/190), 14a/f(1.1%, 2/190), 9h/c(1.1%, 2/190), 15l/f(0.5%, 1/190), 25a/e(0.5%, 1/190), 15i/f(0.5%, 1/190). Conclusion Tp genotypes are diversified in patients with early syphilis in Guangxi, and the genotype 14 d/f is predominant.

Pathologic staining method improvement for detection of pulmonary cryptococcal disease / 肿瘤研究与临床

Objective To investigate the pathological staining method of pulmonary cryptococcal disease. Methods Traditional hexamine silver staining and acidified potassium permanganate solution hexamine silver stained were used to analyze paraffin sections of 51 patients with pulmonary cryptococcal disease. Results Compared with traditional hexamine silver stained, the result of acidified potassium permanganate solution with hexamine silver stained was positive, and the whole process only required 25 minutes with the clear background and cryptococcal cyst capsule structure. Conclusion Compared with the traditional hexamine silver staining, acidification of potassium permanganate solution hexamine silver staining method is simpler,easier,more effective and worthy of wide application.

Application of improved gel electrophoresis to the detection of genotype polymorphisms / 中华实用儿科临床杂志

Objective To explore the traits of improved polyacrylamide gel electrophoresis (PAGE)-ethidium bromide(EB) staining in the detection of genotype polymorphisms.Methods The methods of PAGE-silver staining,agarose gel electrophoresis (AGE)-EB staining and improved PAGE-EB staining in polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect the μ-opioid receptor (OPRM1) A118G genotypes in case group (n =167) infants with intracranial hemorrhage (ICH),and control group(n =163) infants without ICH,to conduct a case study analysis.And the application traits of three methods were compared.Results Genotypes of OPRM1 A118G were GG (169 bp),AG (193 bp,169 bp),AA (193 bp).Both the electrophoresis methods of PAGE-silver staining and PAGE-EB could be used to detect the genotypes of OPRM1 A118G clearly in this study.There was no statistically significant difference between the resolutions of DNA fragments (P =0.884).The first method,which had 13 experiment steps,consuming 4-5 hours,involving 12 kinds of chemical reagents,and the pictures were taken with the camera,was complex,with difficult operation,more time consuming; Compared with the first method,the secondmethod was simple,which had 6 test procedures,consuming 2 hours with 8 reagents,and the pictures were taken by using an automatic gel imager.AGE-EB could not be used to detect genotypes of OPRM1 A118G.Conclusions The method of improved PAGE-EB has the advantages of fast,easy operation,and high resolution,which is worthy of wider application.

Detection of leptospires from infected urine and tissue samples in vitro by modified Fontana silver stain / Detecção de leptospiras na urina e nos tecidos infectados in vitro por impregnação com prata Fontana modificada

INTRODUCTION: The microbiological diagnosis of leptospirosis comprises bacteriological and serological methods. The former ones allow the direct detection of leptospires and are considered presumptive with the exception of culture. Therefore, they constitute invaluable tools for rapid diagnosis, mainly in samples from deceased subjects. OBJECTIVE: To evaluate a modified Fontana silver staining method in experimentally infected samples. MATERIAL AND METHODS: Human and animal (hamster) urine samples were experimentally infected with different strains of Leptospira interrogans sensu lato. Liquid culture medium, leptospira cultures, experimentally infected and non-infected human urine samples, clarified and non-clarified imprints, and clarified and non-clarified suspension smears from tissues of experimentally infected and non-infected hamsters were applied for the ass essment of silver staining. The analytical sensitivity of the assay was compared with dark field microscopy and culture. Other bacterial and fungi species were also used. RESULTS: The modified Fontana silver staining allowed the accurate observation of the well-defined leptospire helical structure. On leptospire cultures from infected human samples, we could observe until (1-10) × 10³ leptospires/ml, higher sensitivity in comparison with direct dark field microscopy and lower in comparison with culture. The best results in tissues were obtained on clarified imprints and non-clarified suspension smears. Morphological and stainable structures compatible with leptospires were not observed in the samples without them. CONCLUSION: This procedure allowed differentiating the characteristic morphology of leptospires. As its application suggests, it consists of a simple and easily conducted procedure with stable reagents.

INTRODUÇÃO: O diagnóstico microbiológico da leptospirose inclui métodos bacteriológicos e sorológicos; os primeiros permitem a detecção direta de leptospiras e, à exceção do cultivo, são considerados como presuntivos, mas constituem ferramentas valiosas para o diagnóstico rápido, principalmente nos falecidos. OBJETIVO: Avaliar a coloração de prata Fontana modificada em amostras experimentalmente infectadas. MATERIAL E MÉTODOS: Urinas humanas e hamsters foram infectados experimentalmente com diferentes cepas de Leptospira interrogans sensu lato. Para a avaliação, foi utilizado meio de cultura líquido, culturas de leptospiras, urinas humanas experimentalmente infectadas e não infectadas, impressões clarificadas e não clarificadas e esfregaços de suspensões clarificadas e não clarificadas a partir de tecidos de hamsters infectados e não infectados para o experimento. A sensibilidade analítica do ensaio foi comparada com a microscopia de campo escuro e de cultura. Outras espécies de bactérias e fungos também foram utilizadas. RESULTADOS: A coloração de prata de Fontana modificada permitiu observar claramente e bem definida a estrutura helicoidal das leptospiras. Nas culturas destas e de urinas humanas infectadas, pôde-se observar até (1-10) × 10³ leptospiras/ml, sensibilidade superior à da microscopia de campo escuro e inferior à da cultura. Os melhores resultados nos tecidos foram obtidos em impressões clarificadas e a partir de esfregaços de suspensões não clarificadas. Estruturas morfológicas e tingidas compatíveis com leptospiras não foram observadas nas amostras livres destas. CONCLUSÃO: Esse procedimento permitiu diferenciar a morfologia característica das leptospiras. É um procedimento simples, fácil de realizar e com reagentes estáveis pelo o que a sua aplicação é sugerida.

Qualitative changes in fetal trabecular meshwork fibers at the human iridocorneal angle / 대한해부학회지

We examined a series of changes that occur in the trabecular meshwork fibers of human eyes during fetal development at 12-30 weeks of gestation. At 12 and 15 weeks, the uveal meshwork was stained black with silver impregnation (indicating the predominance of collagen types III and IV) in the endomysium of the ciliary muscle. At 20 weeks, in combination with Schlemm's canal, a dense fibrous tissue mass corresponding to the trabecular meshwork anlage appeared and was colored black. The anlage was continuous with the corneal endothelium rather than with the ciliary muscle. Until 25 weeks, the trabecular meshwork was identifiable as fragmented fiber bundles that stained red-black, suggesting a mixture of collagen types I, III, and IV. At 30 weeks, half of the ciliary muscle fibers were inserted into the scleral spur and not into the meshwork. Therefore, any contribution of ciliary muscle contraction to the differentiation of the trabecular meshwork would appear to be limited. We hypothesize that an uneven distribution of mechanical stresses in the area of the cornea-sclera junction causes a tear thereby creating Schlemm's canal and is accompanied by a change in the collagen fiber types comprising the meshwork.

A comparison of silver staining protocols for detecting DNA in polyester-backed polyacrylamide gel

Eight silver-staining protocols were applied to detect DNA in polyester-backed gels to select the optimal. Results showed important differences in staining quality and that four methods were well-suited for TGGE gels due to high sensitivity and low background, including the Bassam et al. methods, the manufacturer method and our improved method.

Detection of Treponema pallidum by immunohistochemistry in secondary syphilitic lesions / 中华皮肤科杂志

ObjectiveTo evaluate the diagnostic value of immunohistochemistry(IHC) for the identification and localization of Treponema pallidum(TP) in secondary syphilitic lesions.MethodsSkin tissue specimens were obtained from the lesions of 25 patients with secondary syphilis and 15 patients with dermatoses unrelated to TP infection,followed by fixation and embeding.IHC using a polyclonal antibody against TP and Warthin-Starry (W-S) silver stain were carried out respectively to detect TP in these specimens.Results TP was detected in 80.00％ (20/25) of the specimens by IHC,44.00％ by W-S silver stain (Fisher's Exact Test,P =0.046).Of the 20 IHC-based TP-positive specimens,all harbored TP in the epidermis,11 also in the dermis.The density of TP was associated with the types of skin lesions,and sequentially decreased from condyloma latum to papules,maculopapules and maculae(x2 =15.694,P =0.011 ).Spirochetes were not seen in any of the control lesional specimens.ConclusionsIHC is superior to traditional W-S silver stain for detecting spirochetes in secondary syphilitic lesions,and is of great value to the diagnosis of secondary syphilis.The accurate localization of TP by IHC may facilitate the study on the formation of syphilitic lesions.

Application of the comet assay in erythrocytes of Oreochromis niloticus (Pisces): a methodological comparison

The present study applied the comet assay to erythrocytes of Oreochromis niloticus with the aim of improving protocols to detect DNA damage in these cells, by using two distinct pHs (pH = 12.1 and pH > 13) and evaluating whether there is a correspondence between silver and ethidium bromide staining. Comets were visually examined and, the frequency of cells with and without damage was obtained, as well as the distribution of classes and scores. By using the Kruskal-Wallis test, our results revealed that pH 12.1 is more effective, although both pHs can be used. Our findings also suggest that silver staining can substitute ethidium bromide, an expensive and highly toxic stain that requires specific equipment for examination.

Avaliação da agressividade da lesão de células gigantes periférica da cavidade bucal através de estudo histoquímico-imunohistoquímico / Evaluation of the aggressiveness of peripheral giant cell lesion of the oral cavity by means of a hystochemical-immunohystochemical study

Objetivo: Estudar a agressividade da lesão de células gigantes periférica (LCGP) através de um estudo histoquímico e imunohistoquímico. Metodologia: Foram revisados os arquivos da Faculdade de Odontologia de Araçatuba (FOA-UNESP) e da Faculdade de Odontologia da Universidade de Passo Fundo (FOUPF), tendo sido obtidos 181 casos de LCGP, dos quais 61 preencheram os critérios de elegibilidade. Primeiramente, graduou-se a agressividade das LCGPs, que foram distribuídas em três grupos de estudo com base em achados clínicos e radiográficos. Em seguida, os cortes histológicos das 61 LCGPs, corados por H.E., foram analisados para confirmação diagnóstica. Posteriormente, investigou-se a proliferação celular de 15 casos de LCGPs através de contagem e aferição da área de AgNORs (regiões de organização nucleolar) e avaliação da imunoexpressão dos marcadores PCNA, Ki-67 e p53. Resultados: Não se constatou diferença na área das AgNORs e na expressão do PCNA entre as LCGPs dos três grupos estudados, e o p53 não foi efetivo na imunomarcação das lesões analisadas. Notou-se diferença estatisticamente significativa no número de AgNORs e na expressão do Ki-67 entre os grupos estudados. Conclusão: Os resultados de AgNOR e Ki-67 sugerem que a agressividade clínicoradiográfica das LCGPs pode ser relacionada com sua atividade proliferativa celular.

Purpose: To study the aggressiveness of peripheral giant cell lesion (PGCL) using hystochemical-immunohystochemical analysis. Methods: The archives of the Dental School of Araçatuba (FOA-UNESP) and the University of Passo Fundo Dental School (FOUPF) were searched. A total of 181 PGCL cases were retrieved, and 61 met the study eligibility criteria. Firstly, the PGCL aggressiveness was graded, and the lesions were distributed into three groups based on clinical and radiographic findings. The 61 LCGPs hystopathological samples were colored by H.E. and analyzed for diagnostic confirmation. Afterwards, the cellular proliferation of 15 PGCLs cases was investigated by means of counting and calibrating of AgNORs (nucleolar organizer regions) area and evaluation of the immunoexpression of PCNA, Ki-67, and p53 markers. Results: No difference was found for AgNOR area and PCNA expression among PGCLs of the three studied groups, and p53 was not an effective immunomarker of the analyzed lesions. There was a statistically significant difference of AgNORs number and Ki-67 expression among the three groups. Conclusion: The AgNOR and Ki-67 results suggest that the PGCL clinical-radiographic aggressiveness can be related to its cellular proliferative activity.

Histopathological Changes and Expression of Integrin ?1 of Sternomastoid Muscle in Children with Congenital Muscular Torticollis / 实用儿科临床杂志

Objective To investigate histopathological changes and expression of integrin ?1 of sternomastoid muscle,and probe the mechanism and significance during disease process in congenital muscular torticollis(CMT).Methods Histopathological changes of sternomastoid muscle section stained with HE and Gomori silver staining were observed and the expression of integrein ?1 with immunohistochemistry was detected,and the expressive quantity and distribution with image analysis system was quantitive analyzed.Results 1.With light microscopy observation,the results showed that the fibrous degeneration of sternomastoid muscle could be summed up 2 kinds: A category displayed the myocytes atrophyed,and there were lots of connective tissue hyperplasy around myocytes,and the direction of fibrous arrangement was disordered,meanwhile there were lots of vessels and nervers hyperplasy,and eventually the myocytes shrank back and disappeared.B category displayed that the structure of cross striation or sarcomere disappeared or changed,and myocytes could maintained the outline and the sarcolemma were integrated,and then fibrous pathological changes of myocyte took place,and there were lots of fibroblast-like that had much more enations between fiber bundles.With Gomori silver staining,the major changes of fibrotic sternomastoid muscle showed that there were lots of collagenous fibers hyperplasy.The arrangements of collagenous fibers were disordered in A category and were well-arranged in B ca-tegory.2.With immunohistochemistry,the results showed the expression of integrin ?1 was weak positive in normal control group(125.7?5.167).In diseased groups,the results showed 3 different extents:the expression of integrin ?1 displayed stronger positive in A category myocytes(30.15?6.543),and the level of expression was significantly different from normal controls(P0.05).Conclusions The fibrous pathological changes of sternomastoid muscle are a complicated and gradually process,which may has different mode,and ingetrin ?1 may participated the process of pathological changes.

A method of assessing the thickness of myocardial basement membranes / 中国药理学通报

Aim To observe the thickness changes of cardiomyocyte and microvascular basement membranes(BM)and the expression of collagen Ⅳ in Gk rats of different months and STZ-induced diabetic rats,and to explore the method that operated easily and could assess the thickness of myocardial BM.Methods GK rats of the fourth,fifth,sixth,seventh months,Streptozotocin induced diabetes rats of fifth months and normal Wister rats were respectively divided into different groups with ev eight rats in each group.The myocardial section was stained by methenamine silver and immunohistochemistry was performed for collagen Ⅳ.Left ventricular endocardium was selected as measuring view.Through computer image analysis,the ratio of BM positive staining density(BMPSD)and positive staining density of collagen Ⅳ(CPSD)were calculated separately in all myocardium.Take the ratio to assess the thickness of myocardial BM and the expression of collagen Ⅳ.Results The glycemia,BMPSD and CPSD of the two diabetes rats were higher than those of the normal rats,having highly remarkable difference(P0.05).Conclusion By methenamine silver staining for myocardial section,calculating BMPSD through computer image analysis,can be an index to assess the thickness of myocardial BM.

A Pre-embedding Technique of Nanogold-Silver Staining for Immunocytochemical Localization of Gp100 in Cultured Melanocytes / 대한해부학회지

Nanogold-silver staining is a pre-embedding immunocytochemical technique, making it possible to label antigens with particulate markers at the electron microscopic level. This technique is based on the advancement of gold technology and on the development of silver staining or enhancement. In the present study, the method of Nanogoldsilver staining that includes the use of high concentration of glutaraldehyde and gold toning as well, was used to localize gp100 or a membrane bound antigen in melanosome of the cultured melanocytes. With the HMB45 antibody against gp100, biotinylated secondary antibody and streptavidin-nanogold followed by silver enhancement and gold toning led to highly specific labeling of gold particle over the melanosome compartments. The specificity of labelings obtained with this protocol was confirmed by the control experiment. Omission of the primary antibody led to very low background of labeling. The specific signal that appeared as a collection of gold particles was localized mainly to the peripheral part of melanosome. This finding provides the more detailed nature of gp100 localization in melanosome, which has not yet been shown by the previous immunocytochemical study such as the post-embedding immunoelectron microscopy.

Relationship between the expression of gastrin and PCNA,AgNORs in colorectal cancer tissue / 中国普通外科杂志

Objectives To study the relationship between gastrin expression and proliferating cell nuclear antigen (PCNA),argyrophilia nucleolar organizer regions(AgNORs) in colorectal cancer (CC) tissue. Methods Gastrin and PCNA expression in 48 cases of CC tissue and cancer adjacent mucosa was detected by immunohistochemistry techniques. AgNORs was determined with argyrophilia stain. Results The positive rate of gastrin in CC tissue was 39 58%, that of well differentiated adenocarcinoma was higher than the poorly differentiated and mucinous adenocarcinoma( P

Appraising biofilm of Klebsiella with silver staining method / 中国实用内科杂志

Objective To investigate the reliability of appraising biofilm of Klebsiella with silver staining method.Methods Apply improved flat culture to make biofilm of Klebsiella,adopting silver staining method and scanning electron microscopy to observe and appraise.Results Observation with general optical microscope after silver staining to appraise biofolm is identical with that using scanning electron microscope.Conclusions Silver staining method used to appraise biofilm of Klebsiella is reliable and simple.

An Improved Silver Staining Method / 中国肿瘤生物治疗杂志

A new modification of Merril's silver staining of proteins in sodium dodecylsulfate polyacrylamide gels is introduced.The use of a prolonged period step after fixation and adjusting of oxidant incubation time considerably decrease the background, therefore increase the sensitivity. The detection limit is fivefold lower than original method.

Detection of specific proteins in the aqueous humor in primary open-angle glaucoma

To elucidate the mechanism of increased intraocular pressure in primary open-angle glaucoma (POAG), the protein profiles of aqueous humor obtained from POAG patients were compared with those of cataract patients as a control group. Aqueous humor proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by the ultrasensitive silver staining technique. In 79% of the samples taken from POAG patients, protein bands of 140,000 or 160,000 daltons were stained, but none were stained from cataract patients. The presence of these protein bands revealed statistically significant differences between the two groups. Protein bands of 140,000 or 160,000 daltons were evenly visible at all ages in POAG patients, and the positivity of bands had no correlation with sex or initial intraocular pressure level. It is possible that the ultrastructural changes of the aqueous outflow pathway in POAG may be related to the changes in the aqueous protein, presence of 140,000 or 160,000 daltons protein bands.

STUDIES ON FACTORS INFLUENCING DOT-IGSS FOR ANTIBODIES IN SCHISTOSOMIASIS / 中国血吸虫病防治杂志

By using different blocking and diluting fluids, different developing time and different gold-labeled anti-immunoglobulin (GLA-Ig) which had been preserved in different conditions, the influence on the dot-immunogold-siiver staining (Dot-IGSS) for diagnosis of schis-tosomiasis japonica was studied. The results suggested that using suitable amount of sheep/ bovine serum albumin (BSA) as blocking fluid and calf serum as diluting fluid was a good choice, and 20℃ 20 min was the appropriate time for development. The GLA-Ig without Na3N3 could be preserved in - 20℃ for 2 years if it was not frozen and melted repeatedly.

AMPLIFICATION FRAGMENT LENGTH POLYMORPHISIM OF p33. 4 LOCUS AND ITS APPLICATION TO FORENSIC SCIENCE PRACTICE / 中国法医学杂志

DNA extracted from 100 unrelated Chinese were detected by using the Polymerase Chain Reaction (PCR), mini polyacryl gel vertical electrophorese and silver staining techniques at the p33. 4 locus. Among 100 unrelated individuals, 8 alleles were detected,Foster-Freeman BIOTRAC system indicated that the number of copies of the tandem repeats in different allele was 7, 10 to 15 respectively.There was a rare allele whose copy number was more than 13 but less than 14. The allele fragmehtlength varied from 603 to 1115bp,the allele freqency 0.5 % ~ 53. 5 %,heterozygosity 64 %,DP value84. 5 %. Pedigree analysis indicated that alleles of p33. 4 locus obey Mendel's Laws. Successful typingof various tissues and single hair folicle had confirmed that this technique is suitable for personal identification. In addition, by using Chelex we had established an alterntive method for extracting DNAfrom biological materials,whlch is rapid and easy to perform.