Morphological Quantification of Nuclei and Mitochondria in Serial Block-face Scanning Electron Microscopy Images

Materials and Reagents

1. Adult mouse brain / spinal cord
   
2. 4% Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
   
3. Glutaraldehyde solution (GA) (Sigma-Aldrich, catalog number: G5882 )
   
4. Sodium Cacodylate Buffer (0.4 M, pH 7.2) (Electronic Microscopy Science, catalog number: 11654 )
   
5. Phosphate buffered saline (PBS) (1x Solution, Fisher BioReagents) (Fisher Scientific, catalog number: BP2438-4 )
   
6. Potassium ferricyanide (III) (Sigma-Aldrich, catalog number: 702587 )
   
7. Thiocarbohydrazide (TCH) (Electronic Microscopy Science, catalog number: 21900 )
   
8. Osmium tetroxide 4% solution (Electronic Microscopy Science, catalog number: 19150 )
   
9. Uranyl acetate (UA) (Electron Microscopy Sciences, catalog number: 22400 )
   
10. Lead Nitrate (Crystalline/Certified ACS), Fisher Chemical (Fisher Scientific, catalog number: L62-100 )
    
11. L-aspartic acid solution (Sigma-Aldrich, catalog number: A9256 )
    
12. Ethanol, 200 proof (100%), USP, Decon^TM Labs (Fisher Scientific, catalog number: 07-678-004 )
    
13. Acetone (Electronic Microscopy Science, catalog number: 10010)
    
14. EMbed-812 kit (Electronic Microscopy Sciences, catalog number: 14120 )
    
15. EMS Molded Flat Embedding Mold (Electronic Microscopy Sciences, catalog number: 70905-01 )
    

Equipment

1. Vibratome (Leica, catalog number: Leica VT1000S )
   
2. Zeiss Sigma VP SEM (Zeiss) with Gatan 3View (Gatan)
   
   
   Figure 1. Zeiss Sigma VP SEM (Zeiss) with Gatan 3View (Gatan)
   

Procedure