Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality.
RNA protection
RNA quality
Visium spatial
immunofluorescent staining
methanol fixation
mouse brain transcriptome
spatial transcriptomics
Journal
Frontiers in neuroscience
ISSN: 1662-4548
Titre abrégé: Front Neurosci
Pays: Switzerland
ID NLM: 101478481
Informations de publication
Date de publication:
2023
2023
Historique:
received:
31
03
2023
accepted:
03
05
2023
medline:
5
6
2023
pubmed:
5
6
2023
entrez:
5
6
2023
Statut:
epublish
Résumé
In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols.
Identifiants
pubmed: 37274189
doi: 10.3389/fnins.2023.1198154
pmc: PMC10234422
doi:
Types de publication
Journal Article
Langues
eng
Pagination
1198154Informations de copyright
Copyright © 2023 Hahn, Bens, Kempfer, Reißig, Schmidl and Geis.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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