Analysis of target mRNAs in the fixed-frozen human brain using a modified BaseScope-ISH Assay protocol

M Carla Carisì; Owain W Howell; Alwena H Morgan; Jeffrey S Davies

doi:10.1016/j.xpro.2022.101896

Analysis of target mRNAs in the fixed-frozen human brain using a modified BaseScope-ISH Assay protocol

STAR Protoc. 2022 Dec 16;3(4):101896. doi: 10.1016/j.xpro.2022.101896. Epub 2022 Dec 2.

Authors

M Carla Carisì¹, Owain W Howell², Alwena H Morgan², Jeffrey S Davies³

Affiliations

¹ Institute of Life Science, Swansea University Medical School, Swansea University, Swansea, Wales SA2 8PP, UK. Electronic address: mariacarla.carisi@gmail.com.
² Institute of Life Science, Swansea University Medical School, Swansea University, Swansea, Wales SA2 8PP, UK.
³ Institute of Life Science, Swansea University Medical School, Swansea University, Swansea, Wales SA2 8PP, UK. Electronic address: jeff.s.davies@swansea.ac.uk.

Abstract

We describe a modified BaseScope™ Assay protocol (ACDBio) for RNA in situ hybridization on fixed-frozen human brain tissue. The original protocol caused tissue detachment due to harsh tissue pre-treatment. We therefore optimized it to improve tissue stability while providing high stain quality in fragile post-mortem tissue from aged donors with advanced neurodegeneration. The main changes include two additional fixation steps and modifications to the pre-treatment protocol. We also describe tissue imaging and stain quantification using the open-source QuPath software. For complete details on the use and execution of this protocol, please refer to Hornsby et al. (2020).

Keywords: Health Sciences; In Situ Hybridization; Microscopy; Neuroscience.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aged
Brain* / diagnostic imaging
Coloring Agents
Freezing
Humans
In Situ Hybridization
RNA*
RNA, Messenger / genetics

Substances

RNA, Messenger
RNA
Coloring Agents