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Über den histochemischen und mikrochemischen Nachweis der β-Galactosidase mit 1-Naphthyl-β-galactopyranosid

On the histochemical and microchemical demonstration of β-galactosidase by means of 1-naphthyl-β-galactopyranoside

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Summary

A simultaneous azo coupling method for the intracellular demonstration of acid (hetero-) and neutral β-galactosidase (lactase) in various organs of rats, mice and guinea-pigs is described.

The recommended incubation medium consists of 4.5–9 mg 1-naphthyl-β-galactopyranoside (dissolved in 0.4 ml NN-dimethylformamide) and 0.5–0.8 ml 2% hexazonium-p-rosaniline in 9 ml 0.1 M citrate buffer, pH 5.0 (hetero-β-galactosidase) or 5.5 (lactase).

Among all organs investigated the strongest acid β-galactosidase reaction regularly occurs in the lysosomes of the epididymis, kidney, adrenal, thyroid, preputial and inguinal gland, spleen, colon and chorioid plexus; the neutral β-galactosidase can only be detected in the intestinal brush border exhibiting a moderate activity.

Because hetero-β-galactosidase is a highly soluble enzyme bloc-fixation using glutaraldehyde becomes necessary to achieve a precise intralysosomal localization; for the demonstration of lactase fresh or freeze-dried cryostat sections are suitable. —In the proximal tubule of the rat kidney independent of their concentration the inhibition of acid β-galactosidase following treatment with formol surpasses that of glutaraldehyde. Within the first ten minutes of fixation the enzyme reaches its basis activity. The recovery rate of renal hetero-β-galactosidase considerably increases in the course of washing in hypertonic sugar solution.

In comparison with the indigogenic technique nearly identical results can be obtained with the azo coupling procedure.

Zusammenfassung

Es wird ein simultanes Azokupplungsverfahren zur intrazellulären Darstellung der sauren (Hetero-) und neutralen β-Galactosidase (Lactase) in verschiedenen Organen von Ratte, Maus und Meerschweinchen beschrieben.

Das Inkubationsmedium enthält 4,5–9mg 1-Naphthyl-β-galactopyranosid (gelöst in 0,4ml NN-Dimethylformamid) und 0,5–0,8ml 2% Hexazonium-p-rosanilin in 9 ml 0,1 M Citrat-Puffer, pH 5 (Hetero-β-galactosidase) oder 5,5 (Lactase).

Unter allen Organen reagiert die saure β-Galactosidase am kräftigsten in den Lysosomen von Nebenhoden, Niere, Nebenniere, Schilddrüse, Glandula präputialis und inguinalis, Milz, Colon und Plexus chorioideus; die neutrale β-Galactosidase kommt in mittlerer Aktivität nur im intestinalen Stäbchensaum vor.

Die intralysosomale Darstellung der löslichen Hetero-β-galactosidase erfordert Blockfixation in Glutaraldehyd; die Lactase kann an frischen oder gefriergetrockneten Schnitten untersucht werden. Im proximalen Tubulus der Rattenniere wird die saure β-Galactosidase durch Formol unabhängig von der Konzentration des Fixans verglichen mit Glutaraldehyd stärker gehemmt. Spätestens 10 min nach Beginn der Fixation hat das Enzym seine Basisaktivität erreicht. Spülen in hypertoner Zuckerlösung macht die Inhibition der Hetero-β-galactosidase teilweise rückgängig.

Die mit dem Azokupplungs- und Indigogen-Verfahren gewonnenen Befunde sind weitgehend identisch.

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  1. Anatomisches Institut der Universität, Bundesrepublik Deutschland

    R. Gossrau

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Gossrau, R. Über den histochemischen und mikrochemischen Nachweis der β-Galactosidase mit 1-Naphthyl-β-galactopyranosid. Histochemie 35, 199–218 (1973). https://doi.org/10.1007/BF00305932

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