Effect of Different Staining Methods on Brain Cryosections.

Area of Science:

• Neuroscience
• Molecular Biology
• Genomics

Background:

• Spatial transcriptomic studies of the brain require staining frozen sections to identify cell types.
• Staining methods can impact RNA integrity, a critical limitation for microdissection-based spatial transcriptomics.
• A systematic comparison of staining modalities for brain tissue frozen sections and their effect on RNA quality is lacking.

Purpose of the Study:

• To systematically analyze the effect of four different staining methods on RNA integrity in frozen brain tissue sections.
• To assess the impact of RNA quality differences under various staining conditions on transcriptome sequencing results using RNA-seq.

Main Methods:

• Four distinct staining methods were applied to frozen brain tissue sections.
• RNA integrity was assessed in sections subjected to different staining protocols.
• RNA sequencing (RNA-seq) was performed to evaluate the impact on transcriptome data.

Main Results:

• Hematoxylin and Eosin (HE) staining, a common method, significantly compromises RNA quality in frozen brain sections.
• A newly developed, homemade cresyl violet staining method demonstrated shorter staining times, lower costs, and minimal RNA degradation.
• RNA-seq analysis confirmed that cresyl violet staining resulted in better RNA quality compared to HE staining.

Conclusions:

• The homemade cresyl violet staining method is a viable and advantageous alternative to HE staining for pretreatment of frozen brain tissue sections in transcriptome studies.
• This novel staining technique minimizes RNA degradation, preserving sample quality for downstream analyses like spatial transcriptomics.
• The proposed cresyl violet method has the potential for broad application in brain-related frozen tissue studies and may become a routine step.