Mitigating Fixation Artifacts in Spatial Transcriptomics: Methodological Insights from Human Brain Tissue.

Human brain bank material offers a great potential for studying a wide range of diseases from psychiatric to degenerative disorders, stroke, and pain. Historically, the primary limitation has been the technical usability of long-term formalin fixed and stored tissue samples with inadequate RNA quality. However, new and emerging, highly sensitive techniques now enable the study of the human transcriptome in autopsied brain material. This study aimed to develop a protocol for analysing RNA expression in two distinct cell populations within a spatial context from long-term formalin fixed human hypothalamus samples from the Danish SURVIVE study, using the GeoMx Digital Spatial Profiler from NanoString. RNAscope assays were used to optimize target retrieval, followed by evaluation with QuPath quantification to determine optimal RNA binding conditions. Optimal RNA availability was achieved with baking the slides at 60 °C for 1 h, followed by 30 min of 99 °C heat-induced target retrieval while maintaining tissue integrity. Subsequently, tissue was stained for either microglia or neurons with Iba1 and CRH specific antibodies and analysed in the GeoMx Digital Spatial Profiler. Whole transcriptome probes within the Iba1 and CRH segments were sequenced on the Illumina NovaSeq platform. The novel bioinformatic tool StandR was used for quality control and data analysis. The two cellular segments were compared, and we found 932 differentially expressed genes: 317 upregulated in the CRH segment, and 615 upregulated in the Iba1 segment. This paper presents a detailed workflow and highlights the ability to obtain relevant transcriptomic information from long-term fixed human brain tissue.