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dc.contributor.authorHussain, Sulemanen_US
dc.contributor.authorFredriksen, Ireneen_US
dc.contributor.authorRingsevjen, Håvarden_US
dc.contributor.authorKavalali, Ege T.en_US
dc.contributor.authorDavanger, Svenden_US
dc.date.accessioned2020-06-26T12:02:28Z
dc.date.available2020-06-26T12:02:28Z
dc.date.issued2019
dc.PublishedHussain S, Fredriksen I, Ringsevjen H, Kavalali, Davanger S. Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy. Journal of Neuroscience Methods. 2019;317:1-10eng
dc.identifier.issn0165-0270
dc.identifier.issn1872-678X
dc.identifier.urihttps://hdl.handle.net/1956/23051
dc.description.abstractBackground: Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. New method: To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. Comparison with existing method and results: An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. Conclusion: Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM.en_US
dc.language.isoengeng
dc.publisherElseviereng
dc.rightsAttribution CC BYeng
dc.rights.urihttp://creativecommons.org/licenses/by/4.0eng
dc.titleAntibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopyen_US
dc.typePeer reviewed
dc.typeJournal article
dc.date.updated2020-01-16T10:27:08Z
dc.description.versionpublishedVersionen_US
dc.rights.holderCopyright 2019 The Author(s)
dc.identifier.doihttps://doi.org/10.1016/j.jneumeth.2019.01.015
dc.identifier.cristin1696412
dc.source.journalJournal of Neuroscience Methods


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