Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver.
The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media.(ABSTRACT TRUNCATED AT 250 WORDS)
Griffini
P
P
Department of Animal Biology, University of Pavia, Italy.
Vigorelli
E
E
Bertone
V
V
Freitas
I
I
Van Noorden
C J
CJ
D000818:Animals; D003592:Cytophotometry; D005260:Female; D005782:Gels; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis*; D006651:Histocytochemistry / Q000379:methods*; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D011142:Polyvinyl Alcohol; D012685:Sepharose
D005782:Gels; D011142:Polyvinyl Alcohol; D012685:Sepharose; D007770:L-Lactate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase