Gallyas Silver Impregnation of Myelinated Nerve Fibers

Materials and Reagents

1. Cover slips (Thermo Scientific, Menzel, catalog number: 1011880500)
2. Microscope glass slides (R. Langenbrinck, catalog number: 03-0003)
3. Paraffin
4. 50 ml Falcon tube
5. Mice (postnatal as well as adult brains of any mouse strain are suitable for Gallyas staining)
6. 2-propanol (Roth, catalog number: 6752.4)
7. Acetic acid (Merck Millipore, catalog number: 1000631000) 
8. Acetic anhydride (Acros organics, catalog number: 149490010) 
9. Ammonium nitrate (Merck Millipore, catalog number: 1011880500) 
10. Ethanol (Roth, catalog number: 9065.4) 
11. Eukitt (O. Kindler, catalog number: 4023.1), store at room temperature
12. Formaldehyde 37% (Roth, catalog number: P733.1)
13. KCl
14. KH₂PO₄
15. NaCl
16. NaH₂PO₄
17. Nitric acid (Roth, catalog number: 2616.2)
18. Paraformaldehyde (Serva, catalog number: 31628.02)
19. Pyridine (AppliChem, catalog number: A0776.0500)
20. Silver nitrate (MerckMillipore, catalog number: 1015120025) 
21. Sodium carbonate anhydrous (MerckMillipore, catalog number: 1063920500)
22. Sodium hydroxide (MerckMillipore, catalog number: 1064981000) 
23. Sodium thiosulfate pentahydrate (J.T. Baker, catalog number: 3946-01) 
24. Sucrose (Sigma-Aldrich, catalog number: S9378-5KG)
25. Tungstosilicic acid hydrate (MerckMillipore, catalog number: 1006590025) 
26. Xylene (Otto Fischar, catalog number: 27404)
    
27. 10x PBS stock solution (see Recipes)
28. 4% PFA for tissue (see Recipes)
29. Incubating solution (see Recipes)
30. Physical developing solution (see Recipes)
    1. Solution A
    2. Solution B
    3. Solution C

Equipment

1. Filter unit (Corning, catalog number: 431153)
2. Fume hood (Dueperthal, model: Typ 90)
3. Glassware (rectangular staining dish with glass cover, removable glass slide rack that, dimensions: 91 mm x 71 mm x 60 mm, sold by e.g., VWR)
4. Microscope (Zeiss, model: Axiophot)
5. Microtome (ThermoFisher Scientific, model: HM 430)
6. Paraffin embedding machine (Microm, model: HMP110)
   

Procedure

1. Paraffin embedding and microtome sectioning 
   1. Carefully remove the brain or spinal cord and post-fix it in 4% PFA for tissue for one hour up to overnight at 4 °C in a 50 ml Falcon.
      Note: Prior to paraffin embedding of the brain, mice were subjected to transcardial perfusion.
   2. Embed the brain in paraffin by using an automated embedding machine for best results.
   3. Use the recommended protocol of your embedding machine. If no particular protocol is recommended, use the following protocol: 
      

      Reagent	Duration
      50% ethanol	1 h
      70% ethanol	2x 2 h
      96% ethanol	2x 1 h
      100% ethanol	2x 1 h
      2-propanol	1 h
      xylene	2x 2 h
      paraffin	2x 2 h

   4. Cut 5 µm sections using a microtome and mount sections on microscope glass slides. Air-dry at room temperature (RT). Store at room RT for future use.
      Note: A thickness of 5 µm is optimal for subsequent light microscopy. Mounting on slides is most conservative to the integrity of the sections.
      
      
2. Gallyas silver impregnation (Overview of protocol see Figure 1)
   1. De-paraffinize sections by incubating for 10 min each in xylene, again xylene and 2-propanol/xylene (1:1). Afterwards incubate sections in 100%, 90%, 70%, 50% ethanol and distilled water (dH₂O) for 5 min each.
      Note: Formalin-fixed sections are also suitable for subsequent Gallyas silver staining as described in Pistorio et al., 2005. The conservation of morphology in paraffin sections however is superior to crysections.
   2. To suppress staining of non-myelin tissue, incubate sections in pyridine/acetic anhydride (2:1, e.g., 200 ml of pyridine and 100 ml of acetic anhydride) for 30 min at room temperature (RT).
      Note: Prepare solution fresh, stored solution will turn yellow and needs to be discarded. 
   3. Wash 3 times with dH₂O, for 10 min each.
   4. Submerge sections in incubation solution, microwave for 2 min at 440 W. The incubation solution must not come to boil. The temperature should reach approximately 50 °C. Let it cool down for 10 min at RT until lukewarm. 
   5. Wash sections 3 times in 0.5% acetic acid for 5 min each. 
   6. Develop sections in physical developing solution for 3-15 min. After 1-2 min, start monitoring the reaction under the microscope by placing the sections in 1% acetic acid (= interrupts developing reaction). To determine if staining has reached the desired intensity, compare to the optimal result shown in Figure 2. The color of myelin should be dark brown and should display a good contrast as compared to the background. If too pale, the sections can be exposed once again to the physical developing solution. Repeat until optimal result is reached.
   7. Stop the staining reaction by incubating sections 3 times in 1% acetic acid for 5 min each.
   8. Wash twice in dH₂O for 3 min each.
   9. Incubate sections in 2% sodium thiosulfate for 5 min to fix the stain. 
   10. Wash twice with dH₂O for 5 min each.
   11. Dehydrate section in 50%, 70%, 90%, 100% ethanol for each 5 min, followed by 2-propanol/xylene (1:1), xylene and again xylene for 10 min each.
   12. Mount sections with Eukitt and cover slips.
       Note: Sections, stored in slides boxes at RT, have a shelf life of many years.
   13. Result: Myelin and erythrocytes are stained black, sometimes nuclei are stained too, background is pale yellow (Figure 2).
       
       
       Figure 1. Flowchart depicting individual steps and time specification of deparaffinization, staining, rehydration and mounting procedures
       
       
3. Microscopy
   Images can be acquired using a light microscope at different magnifications (Figure 2).
   
   

   
   Figure 2. Gallyas silver impregnation of a mouse brain. Sagittal view of a 3 month-old mouse brain subjected to the Gallyas protocol described here. Depicted are different regions of the brain: A. hippocampus, B. cortex, C. cerebellum, D. striatum, E. midbrain. CC = corpus callosum, CI = capsula interna, CP = caudoputamen. Scale bar = 100 μm.

Data analysis

The images of the silver-impregnated brain sections can be used as representative images or to compare gross morphological differences in myelination between experimental conditions, mouse strains etc.

Notes

1. Aside from washing steps using water and mounting of sections, carry out the procedure in a chemical fume hood.
2. Wash glassware to be used for silver staining with 1% nitric acid overnight and rinse with water.
3. Do not use metal objects such as spatulas, forceps etc.
4. Do not use metal spatula to weigh silver nitrate.
5. Discard reagents that contain silver in hazardous waste containers.
   

Recipes

1. 10x PBS stock solution 
   

   NaCl	80 g
   KCl	2 g
   NaH2PO4	14.1 g
   KH2PO4	2 g

   Dissolve in 1000 ml dH₂O and adjust pH to 7.3 with NaOH. Store at RT. To prepare 1x PBS, dilute with dH₂O
2. 4% PFA for tissue
   

   Paraformaldehyde	4 g
   Sucrose	4 g

   Dissolve in 100 ml PBS. Can be stored at -80 °C
3. Incubating solution
   

   Ammonium nitrate	1 g
   Silver nitrate	1 g

   Dissolve in 1000 ml dH₂O
   Adjust pH to 7.4-7.6 with NaOH
   Brown precipitate will disappear after shaking. Can be stored for 8-10 weeks at 4 °C 
4. Physical developing solution
   Solution A
   Sodium carbonate anhydrous        50 g
   Dissolve in 1000 ml dH₂O. Can be prepared ahead to procedure and stored at 4 °C for several weeks
   Solution B
   

   Ammonium nitrate	2 g
   Silver nitrate	2 g
   Tungstosilicic acid hydrate	10 g

   Dissolve in 1000 ml dH₂O. Can be prepared ahead to procedure and stored at 4 °C for several weeks.
   Solution C
   

   Ammonium nitrate	2 g
   Silver nitrate	2 g
   Tungstosilicic acid hydrate	10 g
   Formaldehyde (37%)	7 ml

   Dissolve in 1,000 ml dH₂O. Can be prepared ahead to procedure and stored at 4 °C for several weeks
   Final physical developing solution
   50% solution A (100 ml) + 35% solution B (70 ml) + 15% solution C (30 ml)
   Always prepare fresh. Do not store
   

Acknowledgments

This work was supported by Deutsche Forschungsgemeinschaft (DFG, grant STE1117/11-1).

Competing interests

The authors declare no conflict of interest.

Ethics

The use of animals was approved by LANUV (Nordrhein-Westfalen, 84-02.04.2015.A529).

References

1. de Monasterio-Schrader, P., Jahn, O., Tenzer, S., Wichert, S. P., Patzig, J. and Werner, H. B. (2012). Systematic approaches to central nervous system myelin. Cell Mol Life Sci 69(17): 2879-2894. 
2. Erwig, M. S., Patzig, J., Steyer, A. M., Dibaj, P., Heilmann, M., Heilmann, I., Jung, R. B., Kusch, K., Mobius, W., Jahn, O., Nave, K. A. and Werner, H. B. (2019). Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin. Elife 8: e43888. 
3. Funfschilling, U., Supplie, L. M., Mahad, D., Boretius, S., Saab, A. S., Edgar, J., Brinkmann, B. G., Kassmann, C. M., Tzvetanova, I. D., Mobius, W., Diaz, F., Meijer, D., Suter, U., Hamprecht, B., Sereda, M. W., Moraes, C. T., Frahm, J., Goebbels, S. and Nave, K. A. (2012). Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 485(7399): 517-521.
4. Gallyas, F. (1979). Silver staining of myelin by means of physical development. Neurol Res 1(2): 203-209. 
5. Hartline, D. K. and Colman, D. R. (2007). Rapid conduction and the evolution of giant axons and myelinated fibers. Curr Biol 17(1): R29-35. 
6. Hildebrand, C., Remahl, S., Persson, H. and Bjartmar, C. (1993). Myelinated nerve fibres in the CNS. Prog Neurobiol 40(3): 319-384. 
7. Joseph, S., Vingill, S., Jahn, O., Fledrich, R., Werner, H. B., Katona, I., Mobius, W., Mitkovski, M., Huang, Y., Weis, J., Sereda, M. W., Schulz, J. B., Nave, K. A. and Stegmuller, J. (2019). Myelinating glia-specific deletion of Fbxo7 in mice triggers axonal degeneration in the central nervous System together with peripheral neuropathy. J Neurosci 39(28): 5606-5626. 
8. Kidd, G. J., Ohno, N. and Trapp, B. D. (2013). Biology of schwann cells. Handb Clin Neurol 115: 55-79. 
9. Lappe-Siefke, C., Goebbels, S., Gravel, M., Nicksch, E., Lee, J., Braun, P. E., Griffiths, I. R. and Nave, K. A. (2003). Disruption of Cnp1 uncouples oligodendroglial functions in axonal support and myelination. Nat Genet 33(3): 366-374. 
10. Monk, K. R., Feltri, M. L. and Taveggia, C. (2015). New insights on Schwann cell development. Glia 63(8): 1376-1393. 
11. Nave, K. A. (2010). Myelination and the trophic support of long axons. Nat Rev Neurosci 11(4): 275-283. 
12. Nave, K. A. and Werner, H. B. (2014). Myelination of the nervous system: mechanisms and functions. Annu Rev Cell Dev Biol 30: 503-533. 
13. Patzig, J., Kusch, K., Fledrich, R., Eichel, M. A., Luders, K. A., Mobius, W., Sereda, M. W., Nave, K. A., Martini, R. and Werner, H. B. (2016). Proteolipid protein modulates preservation of peripheral axons and premature death when myelin protein zero is lacking. Glia 64(1): 155-174. 
14. Pistorio, A. L., Hendry, S. H. and Wang, X. (2006). A modified technique for high-resolution staining of myelin. J Neurosci Methods 153(1): 135-146. 
15. Simons, M. and Nave, K. A. (2015). Oligodendrocytes: myelination and axonal support. Cold Spring Harb Perspect Biol 8(1): a020479. 
16. Tasaki, I. (1939). The electro-saltatory transmission of the nerve impulse and the effect of narcosis upon the nerve fiber. Amer J Physiol 127: 211-227. 
17. Werner, H. B., Kramer-Albers, E. M., Strenzke, N., Saher, G., Tenzer, S., Ohno-Iwashita, Y., De Monasterio-Schrader, P., Mobius, W., Moser, T., Griffiths, I. R. and Nave, K. A. (2013). A critical role for the cholesterol-associated proteolipids PLP and M6B in myelination of the central nervous system. Glia 61(4): 567-586.