Histochemical demonstration of carbonic anhydrase activity

Summary

Freeze-dried frozen sections are floated on the surface of the freshly prepared incubation mixture (CoSO₄ 1.75 × 10⁻³ M, H₂SO₄ 5.3 × 10⁻² M, NaHCO₃ 1.57 × 10⁻² M and KH₂PO₄ 1.17 to 11.7 × 10⁻³ M; demonstration of weak activity requires high phosphate). A compound containing cobalt and phosphorous precipitates at carbonic anhydrase sites and is converted to CoS. Adequate staining requires only 2–10 minutes of incubation. Actazolamide inhibits the staining reaction in specific concentrations. Actazolamidein vivo, 20 mg/kgi.v. to mice 30 minutes before sacrifice also inhibited the staining. The proportion phosphorous in the specific precipitate increases with KH₂PO₄ of the medium (shown by the addition of⁶⁰Co and³²P). An explanation of the reaction mechanism is given, based on the catalyzed loss of CO₂ in the surface layer. The inclusion of phosphate in the medium makes this modification ofHäusler's method so sensitive that it shows carbonic anhydrase activity in for instance stratum spinosum of the skin.