Orius 832 ccd camera

Peptides (25 µM) were incubated in PBS at 37°C for 24 h under orbital stirring at 300 rpm. After 5 min of water bath sonication (FB15053 ultrasonic bath; Thermo Fisher Scientific Inc., Waltham, MA, US), a 5-µL drop of the peptide solution was deposited onto a carbon-coated copper grid (Ted Pella, Redding, CA, US). Next, 5 µL of 2% uranyl acetate was added and left for 5 min before washing it away with H₂O. The resulting samples were observed with a JEM 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan). Images were acquired using an Orius 832 CCD camera (Gatan Inc., Pleasanton, CA, US).

Larvae were bisected through the swim bladder. Tails were used for genotyping while heads were prepared for transmission electron microscopy. Enucleation of adult eyes was performed after euthanasia in fish water at 4°C. Briefly, all tissues were fixed for 1 hour at room temperature in 0.08 M cacodylate buffer containing 2% paraformaldehyde and 2% glutaraldehyde. Samples then were washed with cacodylate buffer and postfixed in 1% osmium tetroxide for 1 hour at 4°C. Samples were washed again and then dehydrated in a graded methanol series before embedding them in Embed-812/DER736 (Electron Microscopy Sciences; Hatfield, PA, USA), using acetonitrile as a transition solvent. Tails were used for genomic DNA extraction and genotyping as described above. Adult animals were genotyped by fin clips at 2 months old. Semithin sections were made with a Leica EM UC7 ultramicrotome (Leica Microsystems GmbH, Vienna, Austria), stained with toluidine blue, and imaged with a Zeiss AxioImager.Z2 (Carl Zeiss Microscopy, Thornwood, NY, USA). Ultrathin sections were stained with uranyl acetate and lead citrate following standard procedures, and electron microscopy was performed on a Tecnai G2 Spirit BioTWIN 20-120 kV digital electron microscope (FEI Company, Hillsboro, OR, USA). Micrographs were acquired with a Gatan image filter and an Orius 832 CCD Camera (Gatan, Inc., Pleasanton, CA, USA).

Replicas were examined using a 200 kV JEOL 2100 electron microscope equipped with an Orius 832 CCD camera (Gatan) or a OneView CMOS camera (Gatan). Single images were captured with DigitalMicrograph (Gatan). SerialEM was used to generate montage images and acquire tilt series from −60° to +60° at 1° increments^{50 (link)}. Montage blending and tomogram reconstruction were done using the IMOD software suite^{51 (link)}. Total of 13 sets of double-tilt tilt series and 19 montages were acquired, processed, and analyzed.