USE OF LOW-TEMPERATURE AND HIGH K-VITRO TISSUE-PREPARATION FOR X-RAY-MICROANALYSIS( INCUBATION MEDIA FOR IN)

Incubation of tissue slices in physiological buffers gives rise to sig nificant changes in the intracellular ion concentrations, which may di sturb subsequent X-ray microanalysis. In the present study it was atte mpted to design incubation conditions that retain the in vivo conditio ns better. The following variables were investigated: (I) exchange of Na+ in the incubation medium for K+, and exchange of Cl- for the less permeable gluconate anion; (2) incubation at 4 degrees C rather than a t 37 degrees C; and (3) addition of dextran to the incubation medium. Brief exposure (a few seconds) of liver slices to a buffer causes chan ges in the intracellular Na, Cl and K concentrations, depending on the ionic composition of the buffer. Incubation in a normal physiological (high NaCl) buffer at 37 degrees C results in a further increase of N a and Cl and a further decrease in K in liver cells. The changes reach a maximum at 30 min and the concentrations then remain stable through out a 2-h incubation. Incubation in sodium gluconate medium or additio n of dextran to the physiological buffer somewhat reduces the changes in the intracellular ion composition (compared to the standard physiol ogical incubation medium). Incubation in potassium gluconate medium re sults in a decrease in cellular Na and an increase in K. Quantitative morphological studies show that tissue oedema is observed to the same extent in hepatocytes incubated in sodium gluconate, potassium glucona te and physiological buffer containing 10% dextran. However, these buf fers cause significantly less cell oedema than the physiological (high NaCl) buffer. Incubation of liver, cerebral cortex or submandibular g land slices in physiological (high NaCl) solutions at 4 degrees C for 4 h caused a more extensive increase in Nat and decrease in K+ than in cubation at 37 degrees C for 2 h. This suggests inhibition of the Na+, K+-ATPase under these conditions. As compared to incubation at 37 deg rees C for 2 h, tissues incubated in potassium gluconate buffer at 4 d egrees C for 4 h have a cellular K concentration closer to the in situ value. Cholinergic stimulation of tissue slices from cerebral cortex and submandibular gland at room temperature for 1 min shows the best p hysiological response in tissue slices preincubated at 4 degrees C for 4 h in high KCl, potassium gluconate and high NaCl, in this order. Th e response can, however, only be seen, when cholinergic stimulation is carried out in a standard physiological buffer with a high NaCl conce ntration. It is concluded that in vitro storage of tissue for X-ray mi croanalysis is best carried out at 4 degrees C in a solution with a hi gh K+ concentration.