Liquid nitrogen‐based quick freezing: Experiences with bounce‐free delivery of cholinergic nerve terminals to a metal surface

Abstract

The limitations of chemical fixation in permitting the 1:1 quantitative correlations required for convincing ultrastructural explanations of cell biological processes are noted. We describe techniques for obtaining highly reproducible direct quick freezing on the polished surface of pure copper bars dipping into a static dewar of liquid Nz. The importance and the ease of testing and obtaining bounce suppression with commercially available equipment is emphasized. Artefacts caused by tissue damage and bad freezing are illustrated, and a hitherto unrecognized population of presynaptic membrane attached vesicles is described in Torpedine electric organ. Between 15 and 20% of the synaptic vesicles are attached to ca. 30% of the cytoplasmic face of the presynaptic terminal membrane. There is a close correlation between the occurrence of such attachments and the application of electrocyte basal lamina to the external face. We suggest that these vesicles are the 'membrane operators,' 'vesigates,' and 'highly active subpopulation' of vesicles whose existence has been invoked to explain biochemical data in other laboratories. We further speculate that relatively selective Ca pumping by this immediately submembranous population leads to displacement of acetylcholine (ACh) and reloading with newly synthesized ACh. The preferential release of the latter would then be expected.

Aquaporin-4 square array assembly: Opposing actions of M1 and M23 isoforms

“…Individual AQP4 tetramers in rat brain (4) and in CHO cells transfected with M1 and M23 polypeptides were shown by zimuth to the orthogonal lattices; each was selected for identical platinum granularity and image resolution. The effects of shadow angle and azimuth on ability to resolve cross-bridges have been described (23). (Ϸϫ450,000.)…”

“…CHO cells for immunolabeling were immersion-fixed in 1% formaldehyde (22) in SPB for 10 min, rinsed, and stored up to 48 hr in SPB. Tissue sections and CHO cells were infiltrated with 30% glycerol, frozen (23), fractured, and replicated in a JEOL RFD-9010C freeze-fracture device with high-resolution shadowing methods revealing details Ͻ1 nm in nonaveraged images (24). Samples fractured at Ϫ170°C, precoated with 0.8-to 1-nm carbon were shadowed with 1-1.5 nm of platinum at a 60°angle (25).…”

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

“…Rats were chemically fixed by whole-body perfusion (10 min) with 1%, 2% or 4% formaldehyde in Sørensen's phosphate buffer (SPB, pH 7.4); the animals were decapitated; the brains were removed, hand-cut coronal slices were embedded in gelatin; and sections containing SCN were cut into 100-μm-thick slices using a Lancer Vibratome 3000 (Vibratome Company, St. Louis, MO) equipped with a refrigerated stage maintained at 4°C. Samples were infiltrated with 30% glycerol, ultra-rapidly frozen by contact with a −195°C copper "mirror" (Phillips and Boyne, 1984) (Gentleman Jim, Ted Pella, Inc., Redding, CA, USA) or an MF7000 Slam Freezer (Boeckler Instruments, Tucson, AZ), freeze fractured in a JEOL/RFD 9010C freeze-fracture machine (RMC/Boeckler; contact JEOL, Akishima, Japan), rotary coated with 1 nm of carbon, and unidirectionally shadowed with 1.0-1.5 nm of platinum/carbon, followed by 20 nm of carbon (Rash and Yasumura, 1999;Rash et al, 1998Rash et al, , 2004; as modified in Kamasawa et al, 2006). A gold "index" grid containing a droplet of 1.5-2.5% Lexan plastic dissolved in dichloroethane was placed on the frozen surface of the replica, the solvent was evaporated at −35° to −20° C, and the Lexan-stabilized samples were thawed and photo-mapped in a Molecular Dynamics Sarastro 2001 inverted laser scanning confocal microscope (no longer manufactured) or a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Inc., Thornwood, NY).…”

Connexin32-Containing Gap Junctions in Schwann Cells at the Internodal Zone of Partial Myelin Compaction and in Schmidt–Lanterman Incisures

“…Slices of fixed nerves were infiltrated with 30% glycerol in SPB, whereas slices of unfixed tissue were infiltrated with 2.5 M sucrose. Cryoprotected slices were frozen by pneumatically damped rapid contact with a Ϫ185°C copper mirror (Phillips and Boyne, 1984). Samples were freeze-fractured in a JEOL (Peabody, MA) RFD9010 C freeze-fracture device, coated with 0.5-1 nm of carbon (Winkler et al, 2002), shadowed with 1 nm of platinum-carbon, and stabilized with 20 nm of carbon.…”