from WWW http://www.csc.fi/jpr/emt/engelhar/IET/Scandem-98-WWW.htmlpresented at:

SCANDEM-98
The 50th Annual Meeting of the
Scandinavian Society for Electron Microscopy

June 7 - 1, 1998
Espoo, Finland

IMMUNOELECTRON-TOMOGRAPHY (IET) USING MAXIMUM ENTROPY METHOD REVEALS 1.4 nm-GOLD-CONJUGATES IN 3D-RECONSTRUCTIONS OF MCF-7 CELL MICROVILLI

Peter Engelhardt, Juha Ruokolainen*, Fang Zhao, Olli Carpén, and Antti Vaheri
Haartman Institute, P.O.Box 21, FIN-00014 University of Helsinki, Finland, *CSC Scientefic Computing, P.O.Box 405, FIN-02101 Espoo, Finland

INTRODUCTION
In modern EM studies, a resolution of 0.1 nm (1Å) is achievable. However, in studies of biological preparations, especially of thick whole mounts specimens, a resolution better than 1 nm is difficult. In addition to difficulties with preparative techniques, poor resolution is caused mainly by superimposing, due to large depth of focus in EM. Significant improvements in resolution are seen in stereo-pair-studies or in a more modern approach with electron tomography (ET) (1).
Ezrin is a cytoskeletal structural component of cell surface microvilli (2). Here we have used different sized (1.4 nm, 5 nm, 10 nm) gold-conjugates to detect mucin, a cell surface protein, and ezrin in microvilli by immunoelectron microscopic (IEM) analysis. We compared the resolution by immunoelectron tomography (IET) of whole-mounted cells, combined with our maximum-entropy-method (MEM) (1), to conventional IEM techniques. The resolution with IET was superior, as 1.4 nm gold-conjugates could be visualized.

MATERIALS AND METHODS
MCF-7 cells were grown on poly-L-lysin-coated carbon-formvar-Ni-grids, mounted on coverslips, washed with Dulbecco's salt solution (D), prefixed (15-30') in 0.125% glutaraldehyde (GA) in HD (HEPES, 20mM, pH7.4, in D), washed and incubated o/n (overnight) in TXHS (TritonX-100, 0.05%, HEPES, 30mM, pH7.4, NaCl 0.1M, KCl 20mM, MgCl2 5mM, with or without 20mM glycin). The grids were incubated for 1-2 h at room temperature (RT) and at 4^oC o/n with rabbit polyclonal anti-ezrin Ab (1:50 dilution) and mouse anti-mucin mAb (1:25 dilution) in TXHS. After washings in TXHS, the specimens were incubated for 1-2h at RT and 4^oC o/n with gold-conjugated Abs mixtures: 1) goat-anti-rabbit (GAR)-10 nm-gold (Sigma, 1:50 dilution) including GAR-1.4 nm-gold (fluoronanogold, Nanoprobes Inc, 1:25 dilution) and goat-anti-mouse (GAM)-5 nm-gold (Sigma, 1:25 dilution) or2) GAR-10 nm-gold (1:25 dilution) with GAM-5 nm-gold (1:25 dilution) and GAM-1.4 nm-gold (1:25 dilution) in TXHS. After washings, the specimens were postfixed in 1.25% GA (1h-o/n), followed by adding 0.3%OsO4 (Os) for 1-5', in cacodylate (C) buffer (pH7.4), washed in C and treated with 1% tannic acid (TA) in C (1h-o/n), washed in C, dehydrated in methanol (MeOH), stained in (0.002%) uranyl-acetate (UA) in MeOH (30''), washed in MeOH and critical point dried (CPD). For immunofluorescence microscopy, paraformaldehyde fixation was used or GA-autofluorescence was quenched with NaBH4. In immuno-EM