Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus
Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844024025532 |
| _version_ | 1797259888810786816 |
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| author | Adam Humenick M.E. Johnson B.N. Chen M. Wee D.A. Wattchow M. Costa P.G. Dinning S.J.H. Brookes |
| author_facet | Adam Humenick M.E. Johnson B.N. Chen M. Wee D.A. Wattchow M. Costa P.G. Dinning S.J.H. Brookes |
| author_sort | Adam Humenick |
| collection | DOAJ |
| description | Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together. |
| first_indexed | 2024-03-07T21:43:51Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-04-24T23:16:35Z |
| publishDate | 2024-03-01 |
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| spelling | doaj.art-c7f0c985e7d24e8290ac8acc4b0d5ea22024-03-17T07:55:38ZengElsevierHeliyon2405-84402024-03-01105e26522Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexusAdam Humenick0M.E. Johnson1B.N. Chen2M. Wee3D.A. Wattchow4M. Costa5P.G. Dinning6S.J.H. Brookes7Human Physiology, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5042, AustraliaHuman Physiology, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5042, AustraliaHuman Physiology, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5042, AustraliaDepartment of Surgery, Flinders Medical Centre, Bedford Park, SA 5042, AustraliaDepartment of Surgery, Flinders Medical Centre, Bedford Park, SA 5042, AustraliaHuman Physiology, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5042, AustraliaDepartment of Surgery, Flinders Medical Centre, Bedford Park, SA 5042, AustraliaHuman Physiology, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, 5042, Australia; Corresponding author.Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.http://www.sciencedirect.com/science/article/pii/S2405844024025532Fluorescent antibody techniqueIndirectProtein denaturationLipofuscinMyenteric plexusEnteric nervous system |
| spellingShingle | Adam Humenick M.E. Johnson B.N. Chen M. Wee D.A. Wattchow M. Costa P.G. Dinning S.J.H. Brookes Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus Heliyon Fluorescent antibody technique Indirect Protein denaturation Lipofuscin Myenteric plexus Enteric nervous system |
| title | Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus |
| title_full | Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus |
| title_fullStr | Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus |
| title_full_unstemmed | Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus |
| title_short | Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus |
| title_sort | antibody elution with 2 me sds solution uses for multi layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus |
| topic | Fluorescent antibody technique Indirect Protein denaturation Lipofuscin Myenteric plexus Enteric nervous system |
| url | http://www.sciencedirect.com/science/article/pii/S2405844024025532 |
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