Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Abstract

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.

Keywords: Enteric nervous system; Fluorescent antibody technique; Indirect; Lipofuscin; Myenteric plexus; Protein denaturation.

Fig. 1

Prior exposure to 2-ME/SDS has little effect on subsequent immunohistochemical staining. Two specimens from the same patient are shown; a) was pre-treated with 2-ME/SDS; b) was not treated. Both were labelled for immunoreactivity for HuC/D (cyan) and nitric oxide synthase (NOS, white) and photographed with identical exposures. Similar intensity of labelling indicates that 2-ME/SDS does not damage tissue antigens. Scale bars = 50 μm.

Fig. 2

Elution removes both primary and secondary antisera from tissue. Five images of the same ganglion are shown. It was first exposed to anti-NOS primary and AF488-conjugated secondary antisera (NOS – a)). Tissue was then eluted b) which removed all NOS labelling. Incubation with the secondary antiserum alone c) did not restore NOS staining. After eluting the secondary antibody d), incubation with primary then secondary antisera fully restored NOS labelling e). Scale bars = 50 μm.

Fig. 3

Biotin-streptavidin complexes protect labelling from elution. Three specimens from the same patient (a, b, c) were stained with anti-NOS primary followed by biotin-coupled secondary antiserum followed by streptavidin-AF488 tertiary. Specimen a) was eluted after the primary NOS antiserum, then incubated with the secondary and tertiary layers. No labelling was visible (top right), suggesting that the primary antiserum had been eluted. Specimen b) was exposed to the anti-NOS primary (left) followed by biotinylated secondary (left), then eluted with 2-ME/SDS (middle). Incubation with the tertiary (streptavidin-AF488) did not reveal labelling, suggesting that elution had removed both primary and biotinylated secondary from the tissue (right). In c), all 3 antisera were applied in sequence, revealing intense NOS immunoreactivity (left) which was largely unaffected by subsequent 2-ME/SDS (middle). Scale bars = 50 μm.

Fig. 4

A biotinylated axonal tracer, revealed by streptavidin-AF488, was resistant to elution by 2-ME/SDS. Extrinsic axons in human colon were anterogradely labelled with biotinamide and visualised with streptavidin-AF488 (a-left). 2-ME/SDS had little effects on staining intensity (a - right). In contrast, HuC/D labelling of the same ganglion (b - left), visualised with donkey anti-mouse-AF555 secondary (left) was fully removed by the elution (right). Scale bars = 50 μm.

Fig. 5

Labelling with two primary antisera raised in the same species. Two specimens from the same patient were both incubated with primary antiserum raised in mouse to HuC/D, biotinylated secondary and a streptavidin-AF488, revealing immunoreactive myenteric nerve cell bodies (cyan - left column). Specimen a) was then exposed to 2-ME/SDS; while specimen b) was kept in PBS. Both specimens were then incubated with a primary antiserum to NF200 (yellow) also raised in mouse, followed by secondary conjugated to AF555 (middle). In b), every HuC/D-immunoreactive cell body was also labelled in AF555. However in a), some HuC/D-positive cell bodies lacked AF555 labelling entirely (white arrowheads). The 2-ME/SDS may have denatured epitopes of the primary anti-HuC/D and biotinylated secondary so that the NF200 primary and AF555-secondary no longer bound. Scale bars = 50 μm.

Fig. 6

Lipofuscin autofluorescence is reduced after elution. Prior to elution, lipofuscin was visible in all 3 filters a). After elution b) it is largely absent. Images were captured on an IX71 epifluorescence microscope with matched exposures (FITC: 300 ms, CY3: 2s and CY5: 5s). Scale bars = 50 μm.