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 midbrain 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 latency-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 integrated TH counts versus rotarod latency-to-fall indicates a positive slope and modest but significant 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 identification 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.

我们提出了一种强大的、新鲜冷冻的免疫组织化学 (IHC) 方法，无需通过固定和冷冻保存将组织用于 IHC，同时保持长期储存，使用基于 LiCor 的红外 (IR) 量化对免疫反应中脑切片中的 TH 进行敏感评估用于跨研究的定量比较。在 IHC 反应前储存长达 1 年的新鲜冷冻组织中，我们发现我们的方法对 3 个月大的 Sprague-Dawley 大鼠的鱼藤酮治疗高度敏感，并且与 rotarod 潜伏期显着下降相关测量约 2.5 倍。与对照相比，测量的中脑区域显示 TH 信号降低 31%（p < 0.01，t 检验，n = 5）。² = 0.68（p < 0.05，n = 10）。这些结果表明，这种通过 IR 检测快速、基于仪器的量化方法成功地量化了大鼠脑组织中的 TH 水平，而从安乐死到数据输出仅需 5 天。这种方法还允许通过 IHC 识别多个目标，同时在同一动物组织内进行下游分子分析，从而允许每次研究使用更少的动物。