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

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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Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

Journal of Histochemistry & Cytochemistry ◽

10.1177/38.3.1689335 ◽

1990 ◽

Vol 38 (3) ◽

pp. 325-329 ◽

Cited By ~ 19

Author(s):

W van den Brink ◽

C van der Loos ◽

H Volkers ◽

R Lauwen ◽

F van den Berg ◽

...

Keyword(s):

In Situ Hybridization ◽

Silver Staining ◽

Staining Method ◽

Double Staining ◽

Reaction Products ◽

Silver Enhancement ◽

Silver Staining Method ◽

Double Staining Method ◽

Beta Galactosidase

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

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High-sensitivity detection of gold labels by high-angle dark-field STEM imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162028 ◽

1990 ◽

Vol 48 (3) ◽

pp. 892-893 ◽

Cited By ~ 1

Author(s):

Max T. Otten

Keyword(s):

High Sensitivity ◽

Dark Field ◽

Bright Field ◽

Backscattered Electron Imaging ◽

Backscattered Electrons ◽

Average Atomic Number ◽

Highly Sensitive ◽

Backscattered Electron ◽

Electron Imaging ◽

High Sensitivity Detection

Labelling of antibodies with small gold probes is a highly sensitive technique for detecting specific molecules in biological tissue. Larger gold probes are usually well visible in TEM or STEM Bright-Field images of unstained specimens. In stained specimens, however, the contrast of the stain is frequently the same as that of the gold labels, making it virtually impossible to identify the labels, especially when smaller gold labels are used to increase the sensitivity of the immunolabelling technique. TEM or STEM Dark-Field images fare no better (Figs. 1a and 2a), again because of the absence of a clear contrast difference between gold labels and stain.Potentially much more useful is backscattered-electron imaging, since this will show differences in average atomic number which are sufficiently large between the metallic gold and the stains normally used. However, for the thin specimens and at high accelerating voltages of the STEM, the yield of backscattered electrons is very small, resulting in a very weak signal. Consequently, the backscattered-electron signal is often too noisy for detecting small labels, even for large spot sizes.

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Osteoconductive Properties of a Volume-Stable Collagen Matrix in Rat Calvaria Defects: A Pilot Study

Biomedicines ◽

10.3390/biomedicines9070732 ◽

2021 ◽

Vol 9 (7) ◽

pp. 732

Author(s):

Karol Alí Apaza Alccayhuaman ◽

Stefan Tangl ◽

Stéphane Blouin ◽

Markus A. Hartmann ◽

Patrick Heimel ◽

...

Keyword(s):

Extracellular Matrix ◽

Bone Formation ◽

Collagen Matrix ◽

Microcomputed Tomography ◽

Soft Tissue Augmentation ◽

Backscattered Electron Imaging ◽

Quantitative Backscattered Electron Imaging ◽

Rat Calvaria ◽

Backscattered Electron ◽

Electron Imaging

Volume-stable collagen matrices (VSCM) are conductive for the connective tissue upon soft tissue augmentation. Considering that collagen has osteoconductive properties, we have investigated the possibility that the VSCM also consolidates with the newly formed bone. To this end, we covered nine rat calvaria circular defects with a VSCM. After four weeks, histology, histomorphometry, quantitative backscattered electron imaging, and microcomputed tomography were performed. We report that the overall pattern of mineralization inside the VSCM was heterogeneous. Histology revealed, apart from the characteristic woven bone formation, areas of round-shaped hypertrophic chondrocyte-like cells surrounded by a mineralized extracellular matrix. Quantitative backscattered electron imaging confirmed the heterogenous mineralization occurring within the VSCM. Histomorphometry found new bone to be 0.7 mm2 (0.01 min; 2.4 max), similar to the chondrogenic mineralized extracellular matrix with 0.7 mm2 (0.0 min; 4.2 max). Microcomputed tomography showed the overall mineralized tissue in the defect to be 1.6 mm3 (min 0.0; max 13.3). These findings suggest that in a rat cranial defect, VSCM has a limited and heterogeneous capacity to support intramembranous bone formation but may allow the formation of bone via the endochondral route.

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