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Whole Mount Enzyme Histochemistry as a Rapid Screen at Necropsy for Expression of β-Galactosidase (LacZ)–Bearing Transgenes: Considerations for Separating Specific LacZ Activity from Nonspecific (Endogenous) Galactosidase Activity

GEMpath Inc., Cedar City, Utah, USA

Correspondence: Address correspondence to: Dr. Brad Bolon, GEMpath, 2540 N. 400 W., Cedar City, UT, 84720-8400; e-mail: brad{at}gempath.net.

Whole mount enzyme histochemistry to localize lacZ-bearing transgenes (lacZ-WMH) also detects endogenous β-galactosidases. The experiments reported here evaluated lacZ-WMH as a potential tool for transgene expression analysis during high-throughput rodent necropsies. A lacZ-WMH survey of organs from adult, wild-type, male and female mice (C57BL/6, FVB/N) and female rats (Sprague-Dawley) performed at the optimal pH ( 7.0) for bacterial lacZ yielded intense endogenous staining in the gonads, kidney, male accessory sex organs, salivary glands, submucosal glands in the duodenum, and thyroid. Substantial staining occurred in the adrenal cortex, lymph nodes, and linings of the gastrointestinal tract, the urinary bladder and uterus, and (for rat only) in the adenohypophysis, bone marrow, thymus, and trigeminal ganglia. Endogenous galactosidases were distributed similarly in sections of flash-frozen organs used for slide-based lacZ histochemistry (lacZ-SBH) at pH 5.0 (optimal for eukaryotic enzymes). Cerebral neurons were labeled only by lacZ-SBH. At pH 7.4, endogenous but not specific lacZ activity was abolished for lacZ-SBH, while endogenous activity was not halted without reducing specific activity for lacZ-WMH. These data demonstrate that lacZ-WMH is feasible during rodent necropsies for many but not all organs if species-, strain-, and sex-specific divergence in endogenous galactosidase activity is considered and special fixation (3% paraformaldehyde for 3 hours at 4°C) is used.

Key Words: β-galactosidase • lacZ • enzyme histochemistry • whole mount staining • transgenic mouse • background reaction • artifact

Abbreviations: ALP, alkaline phosphatase • CAT, choline acetyltransferase • DMF, N,N-dimethylformamide • EGTA, ethylene glycol-bis[β-aminoethyl ether]-N,N,N,’N’-tetraacetic acid • GFP, green fluorescent protein • HE, hematoxylin and eosin • HEPES, N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid] • lacZ, bacterial (Escherichia coli)-derived β-galactosidase • N A, not applicable • NBF, neutral buffered 10% formalin • PBS, phosphate-buffered saline • PFA, paraformaldehyde • PIPES, piperazine-N,N’-bis[2-ethanesulfonic acid] • RT, room temperature • SBH, slide-based histochemistry • WMH, whole mount histochemistry • WT, wild type • X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactoside

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