Dynamic study of intramembranous particles in human fresh erythrocytes using an “in vitro cryotechnique”

Abstract

For analyses of dynamic ultrastructures of erythrocyte intramembranous particles (IMPs) in situ, a quick-freezing method was used to stabilize the flow behavior of erythrocytes embedded in vitreous ice. Fresh human blood was jetted at various pressures through artificial tubes, in which the flowing erythrocytes were elongated from biconcave discoid shapes to elliptical ones, and quickly frozen in liquid isopentane-propane cryogen (-193 degrees C). They were freeze-fractured using a scalpel in liquid nitrogen, and routinely prepared for replica membranes. Many IMPs were observed on the protoplasmic freeze-fracture face (P-face) of the erythrocyte membranes. Some control erythrocytes under nonflowing or stationary conditions showed IMPs with their random distribution. However, other jetted erythrocytes under flowing conditions showed variously sized IMPs with much closer distribution. They were also arranged into parallel rows in some parts, and aggregated together. This quick-freezing method enabled for the first time the visualization of time-dependent topology and the molecular alteration of IMPs in dynamically flowing erythrocytes.

Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells

“…The IVCT-FS method was employed (Fig. 2i-p) because this original approach avoids the loss of soluble proteins during the immunostaining procedure (Terada et al 2006a(Terada et al , 2006b. This cryotechnique confirmed the specific finding of Dyn2 enrichment in the UCs of the bladder epithelium (Fig.…”

“…Bars 50 μm fixed in 0.25% glutaraldehyde in 0.1 M PB for 30 min, briefly immersed in 10% methanol, and then quickly frozen by the metal contact method with a copper metal block cooled in liquid nitrogen (−196°C; JFD-RFA freezingapparatus, JEOL, Tokyo, Japan). The surface areas of the frozen tissues were freeze-fractured with a scalpel in liquid nitrogen (Terada et al 2006a(Terada et al , 2006b, deeply etched under high vacuum conditions of 2-3×10 −7 Torr at −95°C in a freeze-etching device (FD-3AS, Eiko, Ibaraki, Japan) for 20 min, and rotary-shadowed with platinum metal up to a 2-nm thickness at an angle of 30°and then with carbon at an angle of 90°. The prepared replica membranes were coated with collodion in amyl acetate and treated with household bleach to dissolve tissue components.…”

“…To obtain other bladders by the "in vivo cryotechnique" (S. Ohno et al 1996), the luminal sides of the bladders of anesthetized mice were carefully exposed with a pair of scissors, and a mixture of isopentane-propane cryogen (−193°C) cooled in liquid nitrogen was directly poured over them (Terada et al 2006a(Terada et al , 2006b. The frozen bladders were transferred to liquid nitrogen and then freezesubstituted in acetone containing 2% paraformaldehyde by gradually increasing the temperature (−80°C for 48 h and then −30°C, −10°C, 4°C, and finally room temperature, for 2 h each).…”

Raman Microscopy of Freeze‐dried Mouse Eyeball‐slice in Conjunction with the “in vivo Cryotechnique”

“…1c) in liquid nitrogen, and were transferred into a freeze-etching machine (FD-3AS; Eiko Engineering, Ibaraki, Japan) so that the samples could be freeze-dried under 1-4 3 10 À7 Torr vacuum condition at À958C for 3 h (Fig. 1d) (Terada et al, 2006c). The sections were warmed up to 208C, and taken out of the machine.…”

Application of “in vivo cryotechnique” to detect erythrocyte oxygen saturation in frozen mouse tissues with confocal Raman cryomicroscopy

“…Other specimens in the syringes were quickly frozen by dropping into the IP cryogen ( Fig. 1b-ii), as reported before (Terada et al, 2006b). They were also maintained at dry-ice temperature.…”