Monoclonal antibody, E30-2-1, was raised to antigen D6 that specifically labels acidosomes and DV-II. There was no label on the nascent vacuole membrane or ciliary membranes. This cell had been incubated in 1.1µm latex beads for 3 minutes and fixed without a chase. There is some gold over the cytosol but the nascent vacuole lumen is essentially free of label. TEM taken on 7/12/90 by R. Allen with Zeiss 10A operating at 80kV. Neg. 9,780X. Adapted with permission.
To label membranes inside the cell we used very lightly fixed cells (0.25% glutaraldehyde) that were then rapidly frozen in liquid nitrogen and sectioned later at -100oC. These frozen sections were picked up on drops of methylcellulose and transferred to a Formvar-supported grid. The sections were immunogold labeled (15nm gold) to show the location of the specific antigen inside the cell as well as on the cell surface. Microtome sections prepared at approximately 75nm thickness. The raw negative was scanned with an Epson Perfection V750 Pro and this high resolution image is best used for quantitative analysis. Additional information available at (http://www5.pbrc.hawaii.edu/allen/).
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