scholarly journals Multiplex staining by sequential immunostaining and antibody removal on routine tissue sections

2017 ◽  
Author(s):  
Maddalena Maria Bolognesi ◽  
Marco Manzoni ◽  
Carla Rossana Scalia ◽  
Stefano Zannella ◽  
Francesca Maria Bosisio ◽  
...  
Keyword(s):  
Analysis Software ◽  
In Situ Characterization ◽  
Salt Treatment ◽  
Tissue Sections ◽  
Single Section ◽  
Fluorescent Image ◽  
Spectral Separation ◽  
Secondary Antibodies

ABSTRACTMultiplexing (mplx), labeling for multiple immunostains the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labelled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities.We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than thirty different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Mplx on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory and normal cells.

scholarly journals Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections

2017 ◽  
Vol 65 (8) ◽  
pp. 431-444 ◽  
Author(s):  
Maddalena Maria Bolognesi ◽  
Marco Manzoni ◽  
Carla Rossana Scalia ◽  
Stefano Zannella ◽  
Francesca Maria Bosisio ◽  
...  
Keyword(s):  
Analysis Software ◽  
In Situ Characterization ◽  
Salt Treatment ◽  
Tissue Sections ◽  
Single Section ◽  
Fluorescent Image ◽  
Spectral Separation ◽  
Secondary Antibodies

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

Multiple Iteractive Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

scholarly journals Multiple Iterative Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

scholarly journals Multiple Iterative Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract (What’s new in protocol Version 5: an expanded troubleshooting section, more validated antibodies)Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

scholarly journals Multiple Iterative Labeling by Antibody Neodeposition (MILAN)

2019 ◽  
Author(s):  
Giorgio Cattoretti ◽  
Francesca Maria Bosisio ◽  
Lukas Marcelis ◽  
Maddalena Maria Bolognesi
Keyword(s):  
Image Analysis ◽  
Tissue Section ◽  
Indirect Immunofluorescence ◽  
Analysis Software ◽  
Tissue Sections ◽  
Image Analysis Software ◽  
Single Section ◽  
Fluorescent Image ◽  
Secondary Antibodies

Abstract Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.

scholarly journals In situ ultrastructural characterization of chondroitin sulfate proteoglycans in normal rabbit aorta.

1992 ◽  
Vol 40 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Z S Galis ◽  
M Z Alavi ◽  
S Moore
Keyword(s):  
Chondroitin Sulfate ◽  
Rabbit Aorta ◽  
Specific Information ◽  
In Situ Characterization ◽  
Tissue Sections ◽  
Ultrastructural Characterization ◽  
The Media ◽  
Very High

We used a monoclonal antibody recognizing chondroitin sulfate (CS) to investigate by immunocytochemistry the characteristics displayed in situ by aortic proteoglycans (PG) containing CS side chains. The antibody specifically precipitated metabolically labeled PG from aortic extracts. Anti-CS specificity was also tested directly on tissue sections and was confirmed by the virtual abolition of immunolabeling on those previously digested with CS-specific enzymes. The overall CS-PG distribution assessed by light microscopy after embedding in Lowicryl KM4 by silver-enhanced immunogold recapitulated that obtained on frozen sections with immunoperoxidase. Extracellular concentrations of CS-PG were very high in the innermost regions of aorta and decreased in the media. The reaction was weak and diffuse in the adventitia. By electron microscopy, the detailed labeling of CS-PG discriminated patterns of organization at both the regional and the molecular level and enabled morphometric estimations. In relation to other components of the extracellular matrix, we found that CS-PG and elastin mutually excluded each other, while two types of CS-PG were differently associated with collagen within media or adventitia. The use of high-resolution immunodetection for the in situ characterization of aortic CS-PG could add specific information relevant to many biological processes in which these molecules have been implicated.

Characterization of the glycoconjugates of boar testis and epididymis

Reproduction ◽  
2000 ◽  
pp. 325-335 ◽  
Author(s):  
A Calvo ◽  
LM Pastor ◽  
S Bonet ◽  
E Pinart ◽  
M Ventura
Keyword(s):  
Ricinus Communis ◽  
Lectin Histochemistry ◽  
Apical Part ◽  
Seminiferous Tubules ◽  
In Situ Characterization ◽  
Intense Staining ◽  
Terminal Galactose ◽  
Boar Spermatozoa

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

In Situ Characterization of Soils for Prediction of Stress-Strain Relationship

1983 ◽  
Author(s):  
K. Arulanandan ◽  
Y. Dafalias ◽  
L. R. Herrmann ◽  
A. Anandarajah ◽  
N. Meegoda
Keyword(s):  
In Situ Characterization ◽  
Stress Strain ◽  
Strain Relationship

scholarly journals In-situ characterization of highly reversible phase transformation by synchrotron X-ray Laue microdiffraction

2016 ◽  
Vol 108 (21) ◽  
pp. 211902 ◽  
Author(s):  
Xian Chen ◽  
Nobumichi Tamura ◽  
Alastair MacDowell ◽  
Richard D. James
Keyword(s):  
Phase Transformation ◽  
In Situ Characterization ◽  
X Ray ◽  
Reversible Phase ◽  
Laue Microdiffraction

In Situ Characterization of Catalysis and Electrocatalysis Using APXPS

ACS Catalysis ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 1464-1484 ◽  
Author(s):  
Yong Han ◽  
Hui Zhang ◽  
Yi Yu ◽  
Zhi Liu
Keyword(s):  
In Situ Characterization
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