Fluoro-Jade B Staining for Neuronal Cell Death

Materials and Reagents

1. Superfrost plus Microscope slide (Thermo Fisher Scientific, catalog number: 12-550-17 )
   
2. Cover Glass (Thermo Fisher Scientific, catalog number: 12-545-88 )
   
3. Tissue sample
   
4. Fluoro-Jade B (Merck Millipore Corporation, catalog number: AG310 )
   
5. Paraformaldehyde (Electron Microscopy Science, catalog number: 19210 )
   
6. Potassium permanganate (KMnO₄) (Sigma-Aldrich, catalog number: 223468 )
   
7. DAPI (Life Technologies, catalog number: D3571 )
   Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: D3571”.
   
8. Glacial Acetic acid (CH₃CO₂H) (Sigma-Aldrich, catalog number: A9967 )
   
9. Ethanol
   
10. Xylene (Sigma-Aldrich, catalog number: 534056 )
    
11. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
    
12. Sodium tetraborate decahydrate (Sigma-Aldrich, catalogue number: B9876 )
    
13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
    
14. potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: P3786 )
    
15. potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P9791 )
    
16. 4% paraformaldehyde (see Recipes)
    
17. KPBS (see Recipes)
    
18. 0.2% Fluoro-Jade (see Recipes)
    
19. Fluoro-Jade solution (see Recipes)
    
20. 0.2% DAPI (see Recipes)
    

Equipment

1. Vacuum Desiccators (Thermo Fisher Scientific, catalog number: 08-642-5 )
   
2. Tissue-Tek slide staining set (Electron Microscopy Science, catalog number: 62540-01 )
   
3. 24 slide holder (Electron Microscopy Science, catalog number: 62543-06 )
   
4. Orbital shaker
   
5. Timer
   
6. Slide Warmer
   
7. DPX mounting medium (a mixture of the polystyrene distyrene and the plasticizer dibutylphthalate) (Electron Microscopy Science, catalog number: 13512 )
   

Procedure

1. Mount tissue sections (20-35 μm cut on microtome or cryostat) on Superfrost plus slide and let the slide dry overnight under vacuum.
   
2. Fix the tissue on slide warmer 30 min at 60 °C and/or 20 min in paraformaldehyde 4%.
   
3. Follow by 2 min in KPBS.
   Note: All the following steps are done at room temperature.
   
4. Dehydrate in 50%-70%-100% Ethanol 2 min each.
   
5. Rehydrate by going back in 70%-50% Ethanol and KPBS, 2 min each.
   
6. Incubate in potassium permanganate 0.06% (dilute in water) 5 min at room temperature.
   
7. Rinse in water 1 min.
   
8. Incubate in Fluoro-Jade solution at room temperature for 10 min and gently shake on orbital shaker or by doing several dips during incubation (three dips of few seconds 3 times during incubation). Use opaque cup for the Fluoro-Jade incubation and keep the slide in shelters from light for the rest of the procedure.
   Note: Fluoro-Jade solution and potassium permanganate 0.06% must be prepared fresh.
   
9. Follow by three rinses of 1 min each in water.
   
10. Dry the slides overnight under vacuum at room temperature.
    
11. Clear the slide in Xylene (3 x 2 min).
    
12. Cover slip with DPX and dry 24-48 h under hood before microscope analysis.
    
    
    Figure 1. Example of the setup use for incubation and/or dipping of the slides in different solutions
    

Representative data

Figure 2. FJB-positive neurons in the mouse cerebral cortex following ischemic stroke

Notes

To ensure reproducibility between protocols, use the same method of tissue preparation.

Recipes

1. 4% paraformaldehyde
   Heat 700 ml distilled water at 65 °C
   Add 40 g paraformaldehyde
   5 ml NaOH
   When Paraformaldehyde is completely dissolved add 38 g sodium tetraborate
   Complete at 1 liter with distilled water
   
2. KPBS
   3.81 g Potassium phosphate dibasic
   0.45 g Potassium phosphate monobasic
   8.1 g sodium chloride
   Complete to 1 liter with distilled water
   
3. 0.2% Fluoro-Jade
   Dilute 50 mg Fluoro-Jade B in 25 ml of sterile water and aliquot
   Keep this stock solution at -20 °C in shelters from light
   
4. Fluoro-Jade solution
   Fluoro-Jade B 0.0004%
   Acetic acid 0.1%
   DAPI 0.0001% in water
   
5. 0.2% DAPI
   Dilute 10 mg DAPI in 5 ml of sterile water and aliquot
   Keep this stock solution at 4 °C in shelters from light
   

Acknowledgments

This protocol was adapted from Schmued et al. (1997). This work was supported by grants from the Canadian Institutes for Health Research (CIHR) and the Multiple Sclerosis Scientific Research Foundation of Canada.

References

1. Anderson, K. J., Fugaccia, I. and Scheff, S. W. (2003). Fluoro-Jade B stains quiescent and reactive astrocytes in the rodent spinal cord. J Neurotrauma 20(11): 1223-1231.
   
2. Damjanac, M., Rioux Bilan, A., Barrier, L., Pontcharraud, R., Anne, C., Hugon, J. and Page, G. (2007). Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease. Brain Res 1128(1): 40-49.
   
3. Schmued, L. C., Albertson, C. and Slikker, W., Jr. (1997). Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 751(1): 37-46.