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.

Lysosomal accumulation of SCMAS (subunit c of mitochondrial ATP synthase) in neurons of the mouse model of mucopolysaccharidosis III B

“…Slices were then processed for cryo electron microscopy by the method of Tokuyasu [31][32][33] with modifications [34]. Briefly, slices were incubated overnight in 1.85 M sucrose/ 20% PVP-10/ 50 mM HEPES pH 7.4.…”

In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

“…The resulting vesicle supernatant was fixed with 3% formaldehyde and 0.2% glutaraldehyde for 2 h at room temperature and then pelleted by centrifugation at 100,000 ϫ g for 1 h. The pellet was washed with 0.1 M phosphate buffer, pH 7.4, and free aldehyde groups were quenched by incubation in 50 mM NH 4 Cl for 30 min at room temperature. After three rinses with phosphate buffer, the sample was processed for cryosectioning according to Liou et al (1996). Briefly, the pellet was mixed with 10% gelatin, cooled on ice, cut into small pieces and infiltrated with 2.3 M sucrose overnight at 4°C, frozen in liquid nitrogen on cutting pins, and cryosectioned at Ϫ120°C by using a Leica Ultracut UCT ultramicrotome.…”

“…To localize AP-1 complexes, they were labeled with monoclonal mouse anti-␥-adaptin antibodies followed by goat anti-mouse IgG conjugated to 10-nm colloidal gold (British Biocell International, Cardiff, United Kingdom). Grids were stained and dried as described (Liou et al, 1996) and viewed with a Philips CM10 electron microscope. …”

The Dileucine Motif Within the Tail of MPR46 is Required for Sorting of the Receptor in Endosomes

“…The cells were fixed for 2 h on ice with 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M K-phosphate buffer, pH 7.2, embedded in 10% gelatin and small blocks were immersed in 2.3 M sucrose overnight. Ultrathin cryosections were retrieved from the knives in a 1:1 mixture of 2% methylcellulose and 2.3 M sucrose (22) and labeled with primary antibodies detected with protein A conjugated to gold. In case of monoclonal antibodies, a polyclonal rabbit anti-mouse bridging antibody (Dako, Glostrup, Denmark) was used before detection with protein A-gold (23).…”