Nile Red  别名：Nile Blue A oxazone; Phenoxazone 9; 尼罗红

尼罗红 (Nile Red)是一种疏水荧光染料，可用于脂类及蛋白质等的染色。其荧光强烈依赖于其环境的极性，在水中溶解度差，荧光性差，在疏水环境中的颜色发射从深红色到强黄色不等。在对脂质进行荧光染色时，应选择450nm-500nm之间的黄色激发光，其发射光大于528nm。使用时，尼罗红浓度一般为0.25μg/mL-2.0μg/mL。

使用方法

1. 染色液制备

（1）配置储存液：取适量的尼罗红粉末用无水DMSO充分溶解制备10mM储存液，按照单次用量分装冻存，避免反复冻融，避光保存。
（2）工作液配备：用HHBS或生理缓冲液（pH7）将储存液按1:3000稀释为1×尼罗红工作液，漩涡混匀，应为每个样品准备至少600 μL NR工作液。

【注】：尼罗红的实际染色浓度请参考文献或实验室体系来调整，以上仅供参考。

2. 染色步骤

（1）调整细胞浓度为＞5×10⁴个细胞/毫升。

（2）将待测样品用4% 多聚甲醛固定15分钟，并用1×PBS或蒸馏水洗涤两到三次。（可选）

（3）将细胞与尼罗红工作液于37℃下孵育5-15分钟。

（4）使用1×PBS或蒸馏水适当洗涤后，观察。

（5）荧光显微镜或流式细胞仪检测荧光信号，Ex/Em = 552/636 nm。

小鼠脑组织冷冻切片尼罗红染色

1. Mouse tissue harvesting
Central nervous system tissues, including optic nerves (12 male mice, 15 weeks old), brains (31 mice, 3 female and the rest male mice, 6 were 31 weeks old, the rest were 13–15 weeks old), and dorsal columns (8 male mice, 15 weeks old), were harvested with the protocol varying based on the developmental stage of the mice (neonatal vs. adult). Adult animals were deeply anesthetized by 600 mg/kg of sodium pentobarbital. Intracardiac perfusion was performed with 12 mL of room‐temperature phosphate‐buffered saline (PBS), followed by 12 mL of ice‐cold 4% paraformaldehyde (PFA). Tissues were then postfixed in 4% PFA at 4°C overnight. Cryoprotection was achieved through a sequential sucrose treatment, initially in 20% sucrose, until tissue descent, followed by immersion in 30% sucrose. Brain tissues were then encapsulated in an optimal cutting temperature compound and frozen in isopentane cooled by dry ice. Coronal sections ranging from 20 to 100 μm thickness were cut using a cryostat and collected on VWR Superfrost Plus Micro Slides, ensuring three region‐matched sections per slide. For optic nerves and dorsal columns of adult mice, a similar perfusion and fixation protocol was employed. A 1.2 cm segment of the cervical spine was excised and either fixed as above or transferred for live imaging. Dorsal roots and sciatic nerves were harvested in a similar manner. Five neonatal male mice were euthanized by exposure to 10–15 min of profound hypothermia/hypercarbia using an ice block placed in a CO2 chamber, after which their movement gradually ceased and rigor set in. At this time, a tail pinch test was conducted to confirm unresponsiveness to deep pain, then animals were sacrificed by decapitation, and dorsal roots and sciatic nerves were carefully harvested with minimal delay.