Silver-Stained Golgi Staining for Imaging the Intact Structure of Neurons

doi:10.19894/j.issn.1000-0518.230305

Chinese Journal of Applied Chemistry ›› 2024, Vol. 41 ›› Issue (5): 677-686.DOI: 10.19894/j.issn.1000-0518.230305

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Silver-Stained Golgi Staining for Imaging the Intact Structure of Neurons

Feng ZHOU¹^,², Xiao-Qing CAI¹^,²^,³(), Qiao-Wei TANG⁵, Ying ZHU³^,⁴, Li-Hua WANG³^,⁴, Jun HU³^,⁴

^1.Division of Physical Biology，CAS Key Laboratory of Interfacial Physics and Technology，Shanghai Institute of Applied Physics，Chinese Academy of Sciences，Shanghai 201800，China
^2.University of Chinese Academy of Sciences，Beijing 100049，China
^3.Shanghai Synchrotron Radiation Facility，Shanghai Advanced Research Institute，Chinese Academy of Sciences，Shanghai 201210，China
^4.Institute of Materiobiology，Department of Chemistry，College of Science，Shanghai University，Shanghai 200444，China
^5.Xiangfu Laboratory，Jiashan 314102，China

Received:2023-10-05 Accepted:2024-04-05 Published:2024-05-01 Online:2024-06-03
Contact: Xiao-Qing CAI
About author:caixq@sari.ac.cn
Supported by:
the National Key Research and Development Program of China(2021YFA1200904);Shanghai “Science and Technology Innovation Action Plan” Fundamental Research Project(22JC1401203)

Abstract

Abstract:

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 （K₂Cr₂O₇） and silver nitrate （AgNO₃） 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 K₂Cr₂O₇ was significantly better than that of 20 mg/mL K₂Cr₂O₇ alone， and during the optimization process of the mass concentration of K₂Cr₂O₇， the best staining effect was achieved by 80 mg/mL K₂Cr₂O₇， and the staining effect of 35 mg /mL PFA mixed with 20 mg/mL K₂Cr₂O₇ and 80 mg/mL K₂Cr₂O₇ optimal staining duration ratio was 3：1， the optimal mass concentration of AgNO₃ 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 K₂Cr₂O₇， 40 mg/mL PFA fixation， 3 days 35 mg/mL PFA and 20 mg/mL K₂Cr₂O₇， and 2 days 35 mg/mL PFA and 20 mg/mL K₂Cr₂O₇. PFA and 20 mg/mL K₂Cr₂O₇ mixed solution for 3 days， 80 mg/mL K₂Cr₂O₇ for 1 day and 10 mg/mL AgNO₃ for 5 days had the best effect on the visualization of neuronal morphology in brain tissue. The optimized silver-stained 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 achieve staining of the complete morphological structure of neurons. This optimized method is expected to provide a new staining method for studying the relationship between structural changes of brain neurons and brain diseases.

Key words: Potassium dichromate, Silver nitrate, Neuronal morphology, Silver Golgi staining

CLC Number:

O611

Feng ZHOU, Xiao-Qing CAI, Qiao-Wei TANG, Ying ZHU, Li-Hua WANG, Jun HU. Silver-Stained Golgi Staining for Imaging the Intact Structure of Neurons[J]. Chinese Journal of Applied Chemistry, 2024, 41(5): 677-686.

Figures/Tables 5

Fig.1 Schematic diagram of staining process

Fig.2 Optical microscopy imaging results of PFA-K2Cr2O7 mixture after staining for different time durations

Fig.3 Optical microscopy imaging of K2Cr2O7 stained with different mass concentrations K2Cr2O7. K2Cr2O7 mass concentrations of （A） 20 mg/mL，（B） 40 mg/mL，（C） 60 mg/mL and （D） 80 mg/mL，（E） Sholl analysis of neuronal morphology， red oval dashed box： black deposits， blue oval dashed box： directional nerve fibers， red rectangular dashed box： neuronal structures with soma and dendritic structures， blue rectangular dashed box： vascular structures

Fig. 4 Optical microscope imaging of the tissue under different staining time ratioPFA mixed solution with K2Cr2O7 and K2Cr2O7 solution staining time ratios were 0∶4 （A）， 1∶3 （B）， 2∶2 （C）， 3∶1 （D）， 4∶0 （E），（F） Sholl analysis of neuronal morphology， red oval dashed box： black deposits， blue oval dashed box： incomplete neuronal structures， red rectangular dashed box： neuronal structures with soma and dendritic structures， blue rectangular dashed box： vascular structures

Fig.5 Optical microscopy imaging of stained with different mass concentrations AgNO3. AgNO3 mass concentrations of 5 （A）， 7.5 （B）， 10 （C）， 12.5 （D）， 15 （E） and 20 mg/mL （F），（G） Sholl analysis of neuronal morphology， red oval dashed box： black deposits， blue oval dashed box： incomplete neuronal structures， red rectangular dashed box： neuronal structures with soma and dendritic structures， blue rectangular dashed box： vascular structures

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Silver-Stained Golgi Staining for Imaging the Intact Structure of Neurons

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