Enzyme histochemistry on human placental trophoblasts: the effect of fixation delay on enzyme activity

Abstract

We examined the effect of time delay in tissue fixation on enzyme histochemically detectable enzyme activity and its localization in term human placental trophoblasts. Four placental enzymes, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, and cytochrome c oxidase, were studied. A fixation delay of 15 min did not markedly alter the activity or distribution pattern of the four enzymes, excepted for a slight reduction in cytochrome c oxidase activity and the appearance of dilated endoplasmic reticula positive for glucose-6-phosphatase. A fixation delay of 60 min abolished cytochrome c oxidase activity, but the activities of the other three enzymes remained positive. When the placental tissue was stored at 4 degrees C without cutting for 24 h before fixation, cell degeneration occurred. However, alkaline phosphatase activity was still clearly demonstrable. In enzyme histochemistry, ,,immediate" fixation is superior, but even if this cannot be performed, the placentas, especially when they are from patients with rare disorders, should not be discarded. Observations made here will be useful for clinician's attempting enzyme histochemistry in organs other than the placenta.

Human amniotic epithelial cells are morphologically homogeneous: enzymehistochemical, tracer, and freeze-substitution fixation study

“…There were no findings, such as phagosomal formation, fusion of lysosomes to the phagosomes/plasma membranes, or the appearance of the acid phosphatase to the phagosomal membranes, which we have shown to be the histochemical signs of phagocytosis (Matsubara et al 2000a). CCO labelings were observed in the mitochondria in their intermembrane spaces and intracristal spaces (Figures 2b, b'), which agreed well with our previous observations in other cells (Matsubara et al 1997b;Matsubara et al 2000b). Mitochondria, highlighted by CCO stainings, were present all throughout the cytoplasm, and there were no CCO-negative mitochondria (Figures 2b, b').…”

“…All these enzymes have been proved to play important roles in the placenta, especially in the placental trophoblasts, and we have already enzymehistochemically demonstrated their presence in human placental villous trophoblasts. The fixed specimens were sectioned to 40 µm with a freezing microtome, and the detection of enzyme activity was performed as previously reported by our group: acid phosphatase (cerium method, Robinson and Karnovsky 1983;Matsubara et al 2000b), cytochrome c oxidase (CCO) (DAB method, Seligman et al 1968;Matsubara et al 2000b), Ca ++ ATPase (lead citrate method, Ando et al 1981;Matsubara et al 1987a), alkaline phosphatase (cerium method, Robinson and Karnovsky 1983;Matsubara et al 1987b;, glucose-6-phosphatase (cerium method, Asaka et al 1991;Matsubara et al 1999;, glucose-6-phosphate dehydrogenase (copper ferrocyanide method, Ishibashi et al 1999;Matsubara et al 2001d;Matsubara 2002a;b), ADP-degrading enzymes (lead nitrate method, Matsubara et al 1997a;2001c), NADPH oxidase (cerium method, Briggs et al 1975;, adenylate cyclase (lead citrate method, Matsubara et al 1987c), and guanylate cyclase (lead citrate method, Matsubara et al 1987c). Cytochemical negative controls (ie, substrate omission/inhibitor addition from/to the reaction medium, heating the sections before the cytochemical reaction, etc: described in detail in the corresponding references) were performed to ensure the specific detection of each enzyme activity.…”

Reduced amount of cytochrome c oxidase subunit I messenger RNA in placentas from pregnancies complicated by preeclampsia

“…The decreased expression of cytochrome c oxidase can thus not be ascribed to differences in the gestational age or patient age at delivery. Until now, the expression of cytochrome c oxidase in placental trophoblasts has been shown only for histochemically detectable enzyme activity (17)(18)(19)(20)(21). To our knowledge, this is the first study showing this by using the in situ hybridization technique.…”

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“…Immuno-and enzyme histochemical studies may be hampered by delayed tissue fixation (Benagiano et al 2000;Matsubara et al 2000). Another source for inconsistent findings in immunohistochemistry may be related to the used antibodies as exemplified by a systematic investigation of endothelin antibodies (Wolf et al 2001).…”