A dot-blot-staining method for detecting phosphoproteins with a Phos-tag Aqua fluorescent dye

Silver tetraphenylporphine sulfonate (Ag-TPPS) was synthesized in this laboratory and used as an electron dense stain for elastic tissue (Fig 1). The procedures for the synthesis of tetraphenylporphine sulfonate and the staining method for mature elastic tissue have been described previously.The fine structure of developing elastic tissue was observed in fetal and new born rat aorta using tetraphenylporphine sulfonate, phosphotungstic acid, uranyl acetate and lead citrate. The newly forming elastica consisted of two morphologically distinct components. These were a central amorphous and a peripheral fibrous. The ratio of the central amorphous and the peripheral fibrillar portion changed in favor of the former with increasing age.It was also observed that the staining properties of the two components were entirely different. The peripheral fibrous component stained with uranyl acetate and/or lead citrate while the central amorphous portion demonstrated no affinity for these stains. On the other hand, the central amorphous portion of developing elastic fibers stained vigorously with silver tetraphenylporphine sulfonate, while the fibrillar part did not (compare figs 2, 3, 4). Based upon the above observations it is proposed that developing elastica consists of two components that are morphologically and chemically different.

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A pre-embedding, protein a-gold immunolabelling technique for cytocentrifuged cell monolayers for lm and tem

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142724 ◽

1986 ◽

Vol 44 ◽

pp. 220-221

Author(s):

K. Chien ◽

I.P. Shintaku ◽

A.F. Sassoon ◽

R.L. Van de Velde ◽

R. Heusser

Keyword(s):

Cell Surface ◽

Staining Method ◽

Protein A ◽

Surface Antigens ◽

Cell Suspensions ◽

Cellular Morphology ◽

Cellular Phenotype ◽

Cell Surface Antigens ◽

Cell Monolayers ◽

Diagnostic Histopathology

Identification of cellular phenotype by cell surface antigens in conjunction with ultrastructural analysis of cellular morphology can be a useful tool in the study of biologic processes as well as in diagnostic histopathology. In this abstract, we describe a simple pre-embedding, protein A-gold staining method which is designed for cell suspensions combining the handling convenience of slide-mounted cell monolayers and the ability to evaluate specimen staining specificity prior to EM embedding.

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A combined ultrastructural and gene molecular research for the relationship between human uterine cervical carcinoma and human papillomavirus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015856x ◽

1990 ◽

Vol 48 (3) ◽

pp. 204-205

Author(s):

Kun Lee ◽

Jingyi Si ◽

Ricai Han ◽

Wei Zhang ◽

Bingbing Tan ◽

...

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Intraepithelial Neoplasia ◽

Hpv Infection ◽

Dot Blot ◽

Homologous Sequences ◽

Normal Cervical Epithelium ◽

The Relationship ◽

Chronic Cervicitis

There are more supports for the view that human papillomavirus (HPV) infection might be an etiological factor in the development of cervical cancer when the association of persistent condylomata is considered. Biopsies from 318 cases with squamous cell carcinoma of uterine cervix, 48 with cervical and vulvar condylomata, 14 with cervical intraepithelial neoplasia (CIN), 34 with chronic cervicitis and 24 normal cervical epithelium were collected from 5 geographic regions of China with different cervical cancer mortalities. All specimens were prepared for Dot blot, Southern blot and in situ DNA-DNA hybridizations by using HPV-11, 16, 18 DNA labelled with 32P and 3H as probes to detect viral homologous sequences in samples. Among them, 32 cases with cervical cancer, 27 with condyloma and 10 normal cervical epitheliums were randomly chosen for comparative EM observation. The results showed that: 1), 192 out of 318 (60.4%) cases of cervical cancer were positive for HPV-16 DNA probe (Table I)

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3-Dimensional immunolabeling and in situ hybridization detection using fluorescence photooxidation and intermediate-voltage Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168554 ◽

1994 ◽

Vol 52 ◽

pp. 164-165

Author(s):

Thomas J. Deerinck ◽

Maryann E. Martone ◽

Varda Lev-Ram ◽

David P. L. Green ◽

Roger Y. Tsien ◽

...

Keyword(s):

Laser Scanning ◽

Reactive Oxygen ◽

Confocal Laser Scanning Microscope ◽

Fluorescent Dye ◽

Confocal Laser ◽

3 Dimensional ◽

Voltage Electron ◽

Dimensional Distribution ◽

Electron Microscopes ◽

New Generation

The confocal laser scanning microscope has become a powerful tool in the study of the 3-dimensional distribution of proteins and specific nucleic acid sequences in cells and tissues. This is also proving to be true for a new generation of high contrast intermediate voltage electron microscopes (IVEM). Until recently, the number of labeling techniques that could be employed to allow examination of the same sample with both confocal and IVEM was rather limited. One method that can be used to take full advantage of these two technologies is fluorescence photooxidation. Specimens are labeled by a fluorescent dye and viewed with confocal microscopy followed by fluorescence photooxidation of diaminobenzidine (DAB). In this technique, a fluorescent dye is used to photooxidize DAB into an osmiophilic reaction product that can be subsequently visualized with the electron microscope. The precise reaction mechanism by which the photooxidation occurs is not known but evidence suggests that the radiationless transfer of energy from the excited-state dye molecule undergoing the phenomenon of intersystem crossing leads to the formation of reactive oxygen species such as singlet oxygen. It is this reactive oxygen that is likely crucial in the photooxidation of DAB.

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Enzyme histochemical application and backscattered electron imaging to visualize the distribution of lymphatic capillaries

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161965 ◽

1990 ◽

Vol 48 (3) ◽

pp. 880-881

Author(s):

Seiji Kato

Keyword(s):

Staining Method ◽

Double Staining ◽

Reaction Products ◽

Blood Capillaries ◽

Backscattered Electron Imaging ◽

Histochemical Observation ◽

Backscattered Electron ◽

Double Staining Method ◽

Electron Imaging ◽

Cryostat Sections

Previously, the author repeatedly confirmed the higher 5’-nucleotidase (5’-Nase) and lower alkaline phoaphatase (ALPase) activities in the wall of lymphatic capillaries reacted with the lead-based method relative to those of blood capillaries. The ALPase, on the other hand, is markedly higher in blood capillaries than in lymphatics. On the basis of these enzyme characteristics, the author has developed a 5’-Nase— ALPase double staining method to differentiate small lymphatics from blood capillaries at the level of the light microcsopy. Furthermore, we applied it to histochemical observation of the lead-containing reaction products of 5’-Nase in lymphatics on the same or adjacent cryostat sections using backscattered electron imaging (BEI) in scanning electron microscope (SEM). This paper presents a new applicability of 5’-Nase histochemistry by BEI-SEM to demonstrate the distribution of lymphatic capillaries in tissue blocks.

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