Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 431-444 |
| Number of pages | 14 |
| Journal | Journal of Histochemistry and Cytochemistry |
| Volume | 65 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - Aug 1 2017 |
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Keywords
- antibody removal
- epitope
- immunofluorescence
- multiplex
ASJC Scopus subject areas
- Anatomy
- Histology
Cite this
Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections. / Bolognesi, Maddalena Maria; Manzoni, Marco; Scalia, Carla Rossana; Zannella, Stefano; Bosisio, Francesca Maria; Faretta, Mario; Cattoretti, Giorgio.
In: Journal of Histochemistry and Cytochemistry, Vol. 65, No. 8, 01.08.2017, p. 431-444.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections
AU - Bolognesi, Maddalena Maria
AU - Manzoni, Marco
AU - Scalia, Carla Rossana
AU - Zannella, Stefano
AU - Bosisio, Francesca Maria
AU - Faretta, Mario
AU - Cattoretti, Giorgio
PY - 2017/8/1
Y1 - 2017/8/1
N2 - 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.
AB - 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.
KW - antibody removal
KW - epitope
KW - immunofluorescence
KW - multiplex
UR - http://www.scopus.com/inward/record.url?scp=85026294790&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85026294790&partnerID=8YFLogxK
U2 - 10.1369/0022155417719419
DO - 10.1369/0022155417719419
M3 - Article
C2 - 28692376
AN - SCOPUS:85026294790
VL - 65
SP - 431
EP - 444
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
SN - 0022-1554
IS - 8
ER -