Materials and Reagents

Adult mouse brain
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
Phosphate buffered saline (PBS)
Sucrose
Magnesium chloride, 6-Hydrate (MgCl₂^.6H₂O) (Mallinckrodt Baker, catalog number: 2444-01 )
Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
NP-40 (Sigma-Aldrich, catalog number: I3021 )
Potassium Ferricyanide (ACROS ORGANICS, catalog number: 196785000 )
Potassium Ferrocyanide (Mallinckrodt Baker, catalog number: 6932-04 )
Permount (Fisher Scientific, catalog number: SP15-100 )
O.C.T. compund (Sakura, catalog number: 4583 )
Ethanol (Decon Labs, catalog number: 2716 )
X-gal (American Bioanatycal, catalog number: AB00450-00005 )
1 M MgCl₂ (see Recipes)
10% Sodium deoxycholate (see Recipes)
20% NP-40 (see Recipes)
50 mM Potassium Ferricyanide (see Recipes)
50 mM Potassium Ferrocyanide (see Recipes)
Staining buffer (see Recipes)

Equipment

Cryomold (Sakura, catalog number: 4557 )
Cryostat
37 °C incubator
10-20 Slide Staining Dish with Cover (VWR International, catalog number: 900203 ) or equivalent

Procedure

Mouse is perfused with PBS for 5 min followed by 4% PFA/PBS for another 5 min.
Mouse brain is dissected out and post-fixed with 4% PFA/PBS for 4 h at 4 °C.
Fixed brain is washed with PBS three times and then incubated with 20% sucrose overnight (or until samples sink) and then 30% sucrose overnight.
The brain is mounted on standard cryomold with O.C.T. compound and stored at -80 °C until usage.
40 μm cyro-section is made using cryostat and mounted on the slide-glass.
Slide-glass is washed once with the staining buffer (typically 150 ml for a container) for 10 min at room temperature.
Slide-glass is then incubated with 1 mg/ml X-gal in the staining buffer supplemented with 5 mM Potassium Ferricyanide and 5 mM Potassium Ferrocyanide at 37 °C until color develops.
Notes:
1. This step usually takes 3 h to overnight.
2. Don’t let samples dry.
3. Use 100-200 μl X-gal solutions per slide.
Stained sample is washed with PBS three times (5 min each).
Slide is dehydrated with series of ethanol (50%, 75%, 90%, 100%, 2 min each).
Slide is mounted on permount and can be stored at room temperature.
Sample is ready for imaging at higher magnification (Figure 1).

Figure 1. X-gal staining of Nlk gene trap mouse brain. Strong beta-galactosidase expression of the Nlk gene trap mouse is observed within the Purkinje cell layer from 6-week-old Nlk gene trap mouse cerebellum. MCL, molecular cell layer. PCL, Purkinje cell layer. GCL, granular cell layer.

Notes

The duration of incubation depends on how strongly beta-galactosidase is expressed. Sometimes it requires longer than 2 days of incubation. You can use fresh X-gal solution after overnight incubation.
Wet tissue must be put in the staining container.

Recipes

1 M MgCl₂
Add 10.17 g of MgCl₂ into 40 ml dH₂O
Add dH₂O to 50 ml
Stored at room temperature
10% Sodium deoxycholate
Add 5 g of Sodium deoxycholate into 40 ml dH₂O
Add dH₂O to 50 ml
Stored at room temperature
20% NP-40
Mix 10 ml NP-40 with 40 ml dH₂O
Stored at room temperature
50 mM Potassium Ferricyanide
Add 16.46 g of Potassium Ferricyanide into 800 ml dH₂O
Add dH₂O to 1,000 ml
Stored at 4 °C
Protect from light
50 mM Potassium Ferrocyanide
Add 21.12 g of Potassium Ferricyanide into 800 ml dH₂O
Add dH₂O to 1,000 ml
Stored at 4 °C
Protect from light
Staining buffer
Mix 2 ml of 1 M MgCl₂, 1 ml of 10% sodium deocycholate, 1 ml of 20% NP-40 with 800 ml dH₂O
Add dH₂O to 1,000 ml
Stored at room temperature

Acknowledgments

The authors would like to thank the members of the Lim Lab for feedback on this manuscript. This protocol was adapted from the previously published paper: Ju et al. (2013). This work was supported by the National Institute of Neurological Disorders and Stroke grant NS064146, the Brain and Behavior Research Foundation (Formerly NARSAD), the Alfred P. Sloan Foundation, the National Multiple Sclerosis Society, the Charles H. Hood Foundation, the National Ataxia Foundation, and the Yale Scholar Award Program to J. Lim.

References

Ju, H., Kokubu, H., Todd, T. W., Kahle, J. J., Kim, S., Richman, R., Chirala, K., Orr, H. T., Zoghbi, H. Y. and Lim, J. (2013). Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1. J Neurosci 33(22): 9328-9336.