Effects of NaOH-PIPES buffer used in aldehyde fixative on alkaline phosphatase activity in rat hepatocytes

Abstract

Effects of NaOH-PIPES buffer used as a vehicle for aldehyde fixative on alkaline phosphatase (ALPase) activity demonstrated cyto- and biochemically were compared with those of routinely used cacodylate buffer. The reaction products showing ALPase activity demonstrated ultracytochemically were confined to the bile canalicular membranes when cacodylate buffer (0.1 M) was used. However, when PIPES buffer (0.03 M or 0.1 M) was used, the activity was observed on whole membranes of hepatocytes. The activities of the sinusoidal, lateral and bile canalicular membranes were completely suppressed by an addition of 2.5 mM levamisole. Moreover, the same results were obtained when HEPES or low concentration of cacodylate buffer (0.01 M) was used. Biochemical estimation revealed that much higher activity was retained when PIPES or HEPES buffer was used as compared with that when cacodylate buffer was used. Maximum preservation of ALPase activity was obtained when PIPES buffer was used. Cacodylate buffer showed an inhibitory effect on the hepatic ALPase activity in proportion to the buffer concentration. In conclusion, PIPES buffer preserves the alkaline phosphatase activity much better and is a better vehicle for the aldehyde fixatives in alkaline phosphatase cytochemistry.