Prior to NMR sample loading, the labeled proteins were exchanged into PBS, pH 7.0, 1 mM sodium azide, and 10% D2O by successive concentration and dilution and finally concentrated to 0.5 mM.
The time interval between the successive concentration determinations was reduced to thirty seconds by the use of a continuous flow refractometer.
Weber's and Rosenberg's summaries of the results indicate that biologists can explain initial pattern formation in terms of successive concentration gradients that regulate genes without essential appeal to information concepts.
Sections were then de-waxed in xylene and rehydrated by successive immersion in descending concentrations of alcohol.
The embryos were stained in 1% Alcian Green in 70% ethanol, 1% HCL overnight and then dehydrated using 2 hour steps of successive ethanol concentrations (70 %, 90 and 2×100%).
The bound phospholipids and LPS were eluted using 25 mL steps of the CHCl3/CH3OH/CH3COONH4 mixture [2 3 1 (v/v/v ], with successive CH3COONH4 concentrations of 60, 120, 240, and 480 mM in the aqueous component.
To process for Nissl staining, 40 μm brain sections were mounted onto gelatin-coated slides, were dried for 24 hr, incubated in successive decreasing concentrations of ethanol, followed by 3 min in 0.005% cresyl violet solution containing acetic acid, were dehydrated in ethanol followed by xylenes, and were coverslipped using permount mounting medium.
Following the Xylene baths, the slides were then rehydrated in successive decreasing concentration alcohol baths, from 100% to 95% to 70%, all for 5 minutes and then finally washed three times in PBS 0.1 M for 5 minutes per wash.
Two successive cumulative concentration-response curves for Ca2+ (1 x 10-4 – 1 x 10-2 M) were obtained.
