Cacodylate buffer

N-butyl-β-carboline-3-carboxylate (β-CCB), ethyl 9H-pyrido[3,4-b]indole-3-carboxylate (β-CCE) and 4-ethyl-6,7-dimethoxy-9H-pyrido[3,4-b] indole-3-carboxylic acid methyl ester (DMCM); were all obtained from Tocris Bioscience (Bristol, United Kingdom), isoflurane from PiSa Lab (PiSa, Guadalajara, JAL, México), and Permount mounting medium from Fisher Scientific (FS, Fair Lawn, NJ, United States). Salts, PFA, EB, xylenes and DMSO were acquired from Sigma-Aldrich Co. Glutaraldehyde, cacodylate buffer and osmium tetroxide was from Electron Microscopy Sciences, Hatfield, PA, United States (EMS).

Mice were anesthetized and perfused with 3% glutaraldehyde, 3% formaldehyde in 0.1 M cacodylate buffer (Electron Microscopy Sciences). Cerebellum was cut into small pieces and fixed overnight at 4 °C in 3% glutaraldehyde, 3% formaldehyde in 0.1 M Sorenson’s buffer (Electron Microscopy Sciences). The tissues were then embedded and sectioned at the University of Michigan Histology and Imaging Core. Samples were stained with uranyl acetate/lead citrate and high-resolution images were acquired with a JEOL 1400-plus electron microscope (JEOL).

Organoids were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, #16220) and 2% PFA (Electron Microscopy Sciences, #15710) in 0.1 M sodium cacodylate buffer (Sigma, #20840) overnight at 4°C, washed with 0.1 M cacodylate buffer, and post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences, #19170) for 2 hours at room temperature. Organoids were then dehydrated in a graded ethanol (Sigma, #51976) series (25%, 50%, 75%, and 100%), further dehydrated in propylene oxide (Sigma, #82320) and embedded in Epon resin (SERVA Electrophoresis; glycid ether #21045.01, dodecenylsuccinic acid anhydride #20755.01, methylnadic anhydride #29452.01 and benzyl dimethylamine #14835.01) for 12 h. Semi-thin (0.4 μm) and ultra-thin sections (50 nm) were cut with a Leica EM UC7 ultramicrotome and the latter were collected on formvar-coated single-slot copper grids (Electron Microscopy Sciences, #FF2010-Cu) for imaging. Sections were contrasted with 1% uranyl acetate (Electron Microscopy Sciences, #22400) and lead citrate (Electron Microscopy Sciences, #17800) (8 minutes each) and imaged on a FEI Tecnai Spirit electron microscope (FEI Company) operated at 120 kV using a side-mounted 2K × 2K CCD camera (Veleta, Olympus).

The scutellum of 2 pharate adults, 4 just emerged adults (2 females and 2 males), 4 three day old adults (2 females and 2 males) and 8 ten-day-old adults (4 females and 4 males) of B. oleae were dissected from anaesthetized insects and fixed for 3 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences, Hatfield, England), pH 7.2. The fixed scutelli were repeatedly rinsed in sodium cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in sodium cacodylate buffer (Electron Microscopy Sciences). The samples were then repeatedly washed in the same buffer, dehydrated in ascending ethanol concentrations and finally embedded in an Epon-Araldite resin mixture (Sigma-Aldrich). Afterwards, ultra-thin sections were cut using a Leica EM UC6 ultramicrotome (Leica Microsystem GmbH, Wetzlar, Germany), collected on Parlodion (Spi-Chem, West Chester, PA, USA) coated copper grids, stained with uranyl acetate and lead citrate (Electron Microscopy Sciences, Hatfield, England) and examined using a Technai G2 Spirit BioTwin Transmission Electron Microscope (FEI company, Hillsboro, OR, USA) equipped with a CCD camera Veleta (EMSIS) 2048 × 2040.

pCCTα-RFP was constructed by cloning the CCTα-coding sequence purchased from DNASU plasmid depository (clone ID HsCD00515560) into pmRFP-N1 vector (Clontech). Plasmids pTM-2A-3D, pTM-2B-3D, and pTM-2Amut-3D were described previously [26 (link)]. Plasmid pcDNA3-ACSL3-HA was generously provided by Dr. Joachim Füllekrug, University of Heidelberg, Germany. DNA transfections were performed with Mirus 2020 reagent according to manufacturer’s recommendation. Bodipy 500/510 C4/C9 (a fluorescent long chain fatty analog), Bodipy 493/503 (lipid droplets stain), Alexa-488 azide, and cell click chemistry kit were from Molecular Probes (Thermo Fisher Scientific). Cell culture media and supplements were from Thermo Fisher (GIBCO brand). Propargyl choline was synthesized as described in [58 (link)]. Digitonin was from Calbiochem. Triton-X100 was from Promega. Saponin, DEUP, Orlistat and bafilomycin were from Sigma Aldrich. Formaldehyde, glutaraldehyde, and cacodylate buffer were from Electron Microscopy Sciences. Recombinant universal type I interferon was from PBL Interferon Source.

In a similar manner to a procedure from [21 (link)], the tarsi and tibiae of Ae. albopictus were dissected from anaesthetized insects and fixed for 3 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences, Hatfield, England), pH 7.2. The fixed legs were repeatedly rinsed in sodium cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in sodium cacodylate buffer (Electron Microscopy Sciences). The samples were then repeatedly washed in the same buffer, dehydrated in ascending ethanol concentrations, and finally embedded in an Epon-Araldite resin mixture (Sigma-Aldrich). Afterwards, ultrathin sections were cut using a Leica EM UC6 ultramicrotome (Leica Microsystem GmbH, Wetzlar, Germany), collected on formvar-coated copper grids, and examined using a TEM Philips EM 208 (Philips, Eindhoven, Netherlands).