Preprint Technical Note Version 1 Preserved in Portico This version is not peer-reviewed

Rapid Immunohistological Measurement of Tyrosine Hydroxylase in Rat Midbrain by Near-Infrared Instrument-Based Detection

Version 1 : Received: 24 December 2020 / Approved: 25 December 2020 / Online: 25 December 2020 (07:10:33 CET)

How to cite: Sonne, J.; Seavey, C.; Groshong, J. Rapid Immunohistological Measurement of Tyrosine Hydroxylase in Rat Midbrain by Near-Infrared Instrument-Based Detection. Preprints 2020, 2020120641 (doi: 10.20944/preprints202012.0641.v1). Sonne, J.; Seavey, C.; Groshong, J. Rapid Immunohistological Measurement of Tyrosine Hydroxylase in Rat Midbrain by Near-Infrared Instrument-Based Detection. Preprints 2020, 2020120641 (doi: 10.20944/preprints202012.0641.v1).

Abstract

We present a robust, fresh-frozen approach to immunohistochemistry (IHC), without committing the tissue to IHC via fixation and cryopreservation while maintaining long-term storage, using LiCor-based infrared (IR) quantification for sensitive assessment of TH in immunoreacted mid-brain sections for quantitative comparison across studies. In fresh-frozen tissue stored up to 1 year prior to IHC reaction, we found our method to be highly sensitive to rotenone treatment in 3-month-old Sprague-Dawley rats, and correlated with a significant decline in rotarod laten-cy-to-fall measurement by approximately 2.5 fold. The measured midbrain region revealed a 31% lower TH signal when compared to control (p<0.01 by t test, n=5). Bivariate analysis of in-tegrated TH counts versus rotarod latency-to-fall indicates a positive slope and modest but sig-nificant correlation of R2=0.68 (p<0.05, n=10). These results indicate this rapid, instrument-based quantification method by IR detection successfully quantifies TH levels in rat brain tissue, while taking only 5 days from euthanasia to data output. This approach also allows for the identifica-tion of multiple targets by IHC with the simultaneous performance of downstream molecular analysis within the same animal tissue, allowing for the use of fewer animals per study.

Subject Areas

Neurodegeneration; dopamine; L-DOPA; dopaminergic; DBA; Western; IR; TH; Parkinson disease

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