Materials and Reagents

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

Equipment

Filter unit (Corning, catalog number: 431153)
Fume hood (Dueperthal, model: Typ 90)
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)
Microscope (Zeiss, model: Axiophot)
Microtome (ThermoFisher Scientific, model: HM 430)
Paraffin embedding machine (Microm, model: HMP110)

Procedure

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.
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
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

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

Recipes

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
4% PFA for tissue
Paraformaldehyde
4 g
Sucrose
4 g
Dissolve in 100 ml PBS. Can be stored at -80 °C
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
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).

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