Improved preservation of X-gal reaction product for electron microscopy using hydroxypropyl methacrylate.
In lineage tracing analysis, the beta-galactosidase (beta-gal) gene is a commonly used as a reporter gene because it is relatively stable and highly sensitive in histochemical detection using 5-bromo-4-chloro-3-indolyl-beta-d-galactoside (X-gal). Clear determination of the types and characteristics of labeled cells requires transmission electron microscopic (TEM) examination of their morphology. X-gal staining, which involves the precipitate formed by the reaction between beta-gal and X-gal, is usually recognized as a light blue or green reaction product on light microscopic (LM) examination. However, the standard protocol for TEM preparation weakens the intensity of or results in the loss of X-gal reaction product at the step of substitution of ethanol with Epon using propylene oxide. To solve this problem, we show that hydroxypropyl methacrylate achieves good preservation of X-gal reaction products. The protocol presented here appears to be useful for lineage determination by TEM of all types of X-gal-stained tissues.
Masahira
Noritaka
N
Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan.
Ding
Lei
L
Takebayashi
Hirohide
H
Shimizu
Keiji
K
Ikenaka
Kazuhiro
K
Ono
Katsuhiko
K
D000818:Animals; D019070:Cell Lineage / Q000235:genetics*; D005697:Galactosides; D007211:Indoles; D008689:Methacrylates; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D013116:Spinal Cord / Q000196:embryology / Q000201:enzymology* / Q000648:ultrastructure*; D014021:Tissue Preservation / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D005697:Galactosides; D007211:Indoles; D008689:Methacrylates; D001616:beta-Galactosidase; C032802:hydroxypropyl methacrylate; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside