机构地区:[1]中国科学院上海应用物理研究所,中国科学院微观界面物理与探测重点实验室,上海201800 [2]中国科学院大学,北京100049 [3]中国科学院上海高等研究院,上海光源科学中心,上海201210 [4]上海大学理学院化学系,材料生物学研究所,上海200444 [5]祥符实验室,嘉善314102
出 处:《应用化学》2024年第5期677-686,共10页Chinese Journal of Applied Chemistry
基 金:国家重点研发计划(Nos.2021YFA1200904,2022YFA1603600);上海市“科技创新行动计划”基础研究领域项目(No.22JC1401203)。
摘 要:高尔基染色是神经元树突和树突棘可视化的经典方法,目前仍被广泛用于神经元形态可视化。银高尔基染色目前存在易产生黑色沉淀、染色神经元形态结构不完整等问题。对重铬酸钾(K_(2)Cr_(2)O_(7))和硝酸银(AgNO_(3))如何影响银染高尔基染色方法的染色效果进行了研究,通过研究染色液组成成分重铬酸钾、硝酸银质量浓度变化和组分的不同作用方式,实现了对银染高尔基染色方法的优化。结果表明,35 mg/mL多聚甲醛(Paraformaldehyde,PFA)和20 mg/mL K_(2)Cr_(2)O_(7)混合液染色效果要明显优于20 mg/mL K_(2)Cr_(2)O_(7)单独染色效果,在K_(2)Cr_(2)O_(7)的质量浓度优化过程中,80 mg/mL K_(2)Cr_(2)O_(7)的染色效果最佳,35 mg/mL PFA与20 mg/mL K_(2)Cr_(2)O_(7)混合溶液和80 mg/mL K_(2)Cr_(2)O_(7)最佳染色时长比为3∶1,染色过程中AgNO_(3)的最佳质量浓度为10 mg/mL,在最终的染色液组成成分以及作用方式中40 mg/mL PFA固定2 d、35 mg/mL PFA和20 mg/mL K_(2)Cr_(2)O_(7)混合溶液处理3 d、80 mg/mL K_(2)Cr_(2)O_(7)处理1 d和10 mg/mL AgNO_(3)处理5 d对脑组织神经元形态的可视化具有最佳效果。优化后的银染高尔基染色方法可实现对神经元的完整形态结构染色,同时在染色过程也将产生更少的成像杂质沉淀噪点。该工作将提供一种新的高尔基改良染色方法,实现神经元完整形态结构的染色,有望为研究脑神经元结构变化与脑疾病之间的关系提供新的染色方法。Golgi staining is a classical method for visualization of neuronal dendrites and dendritic spines and is still widely used for neuronal morphology visualization.Silver Golgi staining currently suffers from problems such as the tendency to produce black precipitation and incomplete morphological structure of stained neurons.We investigated how potassium dichromate(K_(2)Cr_(2)O_(7))and silver nitrate(AgNO_(3))affect the staining effect of the silver Golgi staining method,and realized the optimization of the silver Golgi staining method by studying the change of the mass concentration of the staining solution constituents,potassium dichromate,and silver nitrate,and the different modes of action of the components.The results showed that:the staining effect of the mixture of 35 mg/mL paraformaldehyde(PFA)and 20 mg/mL K_(2)Cr_(2)O_(7)was significantly better than that of 20 mg/mL K_(2)Cr_(2)O_(7)alone,and during the optimization process of the mass concentration of K_(2)Cr_(2)O_(7),the best staining effect was achieved by 80 mg/mL K_(2)Cr_(2)O_(7),and the staining effect of 35 mg/mL PFA mixed with 20 mg/mL K_(2)Cr_(2)O_(7)and 80 mg/mL K_(2)Cr_(2)O_(7)optimal staining duration ratio was 3:1,the optimal mass concentration of AgNO_(3)during the staining process was 10 mg/mL,and in the final staining solution composition and the mode of action of 2 days 40 mg/mL PFA fixation,3 days 35 mg/mL PFA and 20 mg/mL K_(2)Cr_(2)O_(7),40 mg/mL PFA fixation,3 days 35 mg/mL PFA and 20 mg/mL K_(2)Cr_(2)O_(7),and 2 days 35 mg/mL PFA and 20 mg/mL K_(2)Cr_(2)O_(7).PFA and 20 mg/mL K_(2)Cr_(2)O_(7)mixed solution for 3 days,80 mg/mL K_(2)Cr_(2)O_(7)for 1 day and 10 mg/mL AgNO_(3)for 5 days had the best effect on the visualization of neuronal morphology in brain tissue.The optimized silverstained Golgi staining method will achieve complete morphological structural staining of neurons,and will also produce less imaging impurity precipitation noise during the staining process.This work will provide a new modified Golgi staining method to achiev
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