Antibody elution with 2-me/SDS solution : Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus
© 2024 The Authors..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:10 |
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| Enthalten in: |
Heliyon - 10(2024), 5 vom: 15. März, Seite e26522 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Humenick, Adam [VerfasserIn] |
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| Links: |
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| Themen: |
Enteric nervous system |
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| Anmerkungen: |
Date Revised 05.03.2024 published: Electronic-eCollection Citation Status PubMed-not-MEDLINE |
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| doi: |
10.1016/j.heliyon.2024.e26522 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM369241487 |
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| 245 | 1 | 0 | |a Antibody elution with 2-me/SDS solution |b Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus |
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| 520 | |a © 2024 The Authors. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Enteric nervous system | |
| 650 | 4 | |a Fluorescent antibody technique | |
| 650 | 4 | |a Indirect | |
| 650 | 4 | |a Lipofuscin | |
| 650 | 4 | |a Myenteric plexus | |
| 650 | 4 | |a Protein denaturation | |
| 700 | 1 | |a Johnson, M E |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, B N |e verfasserin |4 aut | |
| 700 | 1 | |a Wee, M |e verfasserin |4 aut | |
| 700 | 1 | |a Wattchow, D A |e verfasserin |4 aut | |
| 700 | 1 | |a Costa, M |e verfasserin |4 aut | |
| 700 | 1 | |a Dinning, P G |e verfasserin |4 aut | |
| 700 | 1 | |a Brookes, S J H |e verfasserin |4 aut | |
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