Analysis of galactocerebrosidase activity in the mouse brain by a new histological staining method.
Gene therapy of galactocerebrosidase (GALC) deficient mice (Twitcher mutants) requires a fast and sensitive assay to detect transduced cells in vitro and in vivo. We have developed a new rapid histochemical method that specifically detects GALC activity in situ in neural cells using 5-Br-3Cl-beta-galactopiranoside (X-Gal) in the presence of taurodeoxycholic and oleic acids to enhance suspension of the substrate at low pH. Using this method, we observed robust X-Gal staining in diverse neuronal populations and interfascicular oligodendrocytes in sections from normal mouse brain. In contrast, sections of Twitcher brain did not show a specific staining pattern in neurons or glial cells. The availability of this new sensitive and rapid in situ detection assay is fundamental for the follow-up of Twitcher mice under gene or cellular therapies to correct central GALC deficiency.
Dolcetta
D
D
San Raffaele Telethon Institute for Gene Therapy, Milano, Italy.
Perani
L
L
Givogri
M I
MI
Galbiati
F
F
Orlacchio
A
A
Martino
S
S
Roncarolo
M G
MG
Bongarzone
E
E
D000818:Animals; D001921:Brain / Q000201:enzymology*; D004789:Enzyme Activation; D005698:Galactosylceramidase / Q000032:analysis* / Q000172:deficiency / Q000378:metabolism*; D006651:Histocytochemistry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D008818:Mice, Neurologic Mutants; D009457:Neuroglia / Q000201:enzymology; D009474:Neurons / Q000201:enzymology; D013194:Staining and Labeling / Q000379:methods*
D005698:Galactosylceramidase