Improved preservation of X-gal reaction product for electron microscopy using hydroxypropyl methacrylate

In lineage tracing analysis, the beta-galactosidase (β-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-β-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 β-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.