Improving structural integrity of cryosections for immunogold labeling

Abstract

Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution.

Analysis of primary cilia in the developing mouse brain

“…The first method involves a protocol similar to light microscopic staining and therefore yields comparable labeling intensities, but results in a poor structural preservation due to the need of permeabilization for antibody penetration (Humbel, de Jong, Muller, & Verkleij, 1998). The latter method (on-section labeling) can be applied either on plastic-embedded samples (as described elsewhere (Schwarz & Humbel, 2014)) or on sucrose-embedded Tokuyasu cryosections (Liou, Geuze, & Slot, 1996;Tokuyasu, 1973). Labeling of (methacrylate) plastic sections is convenient, even for serial sections, but has the limitation that many epitopes (up to 90%) are destroyed or masked by the resin embedding.…”

Colocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in cells of the blood-ocular barrier in the mouse eye

“…1996b). In short, grids were floated on PBS containing 5% normal goat serum and 1% gelatin for 10 min followed by incubation with rabbit anti-ZO-1 antibody for 1.5 h, and affinity-purified goat anti-rabbit IgG (Jackson Immunoresearch)-colloidal gold (diameter 10 nm) conjugate [prepared according to DeMey (1984) and Slot and Geuze (1985)] for 1 h. They were subsequently washed with PBS and refixed with 2% glutaraldehyde, after which the specimens were treated with uranyl acetate and embedded in 1.8% methylcellulose-0.5% uranyl acetate (Liou et al 1996). Specimens were observed with a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan).…”

Advanced glycation end products impair the scavenger function of rat hepatic sinusoidal endothelial cells

“…Sections were obtained from frozen pellets of SEC using an Ultracut S Ultramicrotome with an FCS cryo chamber (Leica, Vienna, Austria), and sectioned with a diamond knife (Drukker International, Cuijk, The Netherlands) at -90°C. The sections were retrieved in 2.3 mol/l sucrose and methylcellulose (50/50) [46], and transferred to carbon-coated grids. Immune cytochemical labelling was done as described [47].…”