Tecnai t12 microscope

Mouse pancreatic islets were isolated as previously described from 16 to 18-week-old male and female mice, tabulated separately. The islets were subsequently harvested in 1% NP-40 lysis buffer for evaluation of protein content for immunoblotting. For TEM, freshly isolated mouse pancreatic islets were placed immediately in 0.1 M cacodylate-buffer, pH 7.3, 2.5% glutaraldehyde, and 4% paraformaldehyde. After fixation for 2 h RT, the specimens were stored at 4 °C and later post-fixed for 1 h in 0.1 M cacodylate buffer, 1% OsO₄. After rinsing in distilled water, the specimens were en bloc stained for 1 h in 1% uranyl acetate in distilled water. The specimens were dehydrated as follows: 30%, 50%, 60%, 70%, 80%, and 95% ethanol for 10 min each, then three changes of 100% dry ethanol for 10 min each, followed by transfer into 100% propylene oxide (PPO) for 10 min. For resin infiltration, the specimens were transferred to 2:1 PPO/Epon 812 resin for 2 h, then to 1:1 PPO/resin overnight, then to 1:2 PPO/resin for 2 h, and to 100% resin for 3–4 h RT. The specimens were transferred to resin molds and placed at 60 °C for 48 h. Ultrathin (70-nm) sections were cut with a Leica Ultracut, picked up on copper grids, post-stained with lead citrate and viewed with a FEI Tecnai T12 microscope operating at 120 KV. Mitochondrial numbers were counted in a blinded fashion.

To determine the sample quality and success of the Fab binding, purified protein was diluted to 0.01 mg/ml (for PfCRT without Fab) or 0.005 mg/ml (for PfCRT with Fab) and applied onto copper grids (Ted Pella). These grids were overlaid by a thin (∼1.5 nm) layer of continuous carbon that had been plasma-cleaned (Gatan Solarus) for 30 s using a mixture of H₂ and O₂. Thereafter, filter paper (Whatman 4) was used to remove the protein solution. 3 μl of 2% uranyl formate was then added and immediately removed by absorbing with filter paper – this was repeated seven times. The grid was imaged on either a Tecnai T12 microscope (FEI) (for PfCRT without Fab) or a Tecnai TF20 microscope (FEI) (for PfCRT with Fab). Both microscopes were equipped with a Tietz F416 CCD camera (Tietz) at 1.23 Å or 1.10 Å per pixel respectively, using the Leginon software package⁴⁰ . 166 and 87 images were collected respectively and were processed using the Appion software package⁴¹ to obtain 2D classes with Relion 2.1^{42 ,43} . The micrographs showed good particle dispersion. 2D class averages showed that the Fab addition resulted in a clear fiducial for particle alignment (Extended Data Fig. 2).