Journal: Frontiers in Plant Science

Article Title: Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography

doi: 10.3389/fpls.2023.1261180

Figure Lengend Snippet: Protoplasts vitrification by plunge-freezing and Correlative Light and Electron Microscopy (Steps 19 - 29 and 40 - 47) (A) , Diagram showing the steps to follow after protoplast isolation: Plunge freezing procedure and observing the EM grids containing the root protoplasts under a cryo-CLEM, (B-D) , Cryo-Fluorescence Microscopy of a grid containing root-protoplasts from Arabidopsis thaliana’s RAE1-GFP transgenic line. Brightfield: field of bright light; GFP: Green fluorescence signal (green); Merge shows the overlay of the brightfield and GFP signal.

Article Snippet: FB0010) - KOH (Millipore Sigma, cat. No. 105033) - 0.2 M 4-morpholineethanesulfonic acid (MES, pH 5.7; FisherBiotech, cat. no. BP300) - 0.8 M mannitol (Acros, cat. no.125340010) - 1 M CaCl 2 (Bio Basic, cat. no. CT1330) - 2 M KCl (Fisher scientific, cat. no. P330-500) - 2 M MgCl 2 (Fisher Scientific, cat. no.M-20944) - 154 mM NaCl (Fisher Scientific, cat. no. S271) - Tris Base (Millipore Sigma, cat. no. 648310) - HCl (Millipore Sigma, cat. no. 258148) - β-Mercaptoethanol (Fisher Scientific, cat. no. BP176-100) - BSA (Roche, cat. no. 10 735 086 001) - Cellulase from ( Trichoderma sp., Sigma-Aldrich, cat. no. C0615-1G) - Pectinase from ( Aspergillus niger , Sigma-Aldrich, cat. no. P4716) - Concanavalin A (Sigma Aldrich, cat. no. C2010) - Enzyme solution (see REAGENT SETUP) - W5 solution (see REAGENT SETUP) BIOLOGICAL MATERIALS - Arabidopsis accessions: Col-0, RAE1-GFP ( ) EQUIPMENT - Square Petri Dishes (Fisher Scientific, cat no. FB0875711A) - Sorvall Legend XFR Centrifuge (Thermo Fisher Scientific, cat. no. 75004541) - 0.2-μm Syringe filter – Sterile (Fisher Scientific, cat. no. 09-719C) - Sterile Single Use Carbon Blades (Lance, cat. no. 1500321) - Petri dish with clear lid (Fisher Scientific, cat. no. FB0875712) - Micropore (3M, cat. no. 1530-1) - Sterile Cell Strainer 70 μm Nylon Mesh (Fisher Scientific, cat. no. 22-363-548) - Improved Neubauer 0.1-mm-deep hemacytometer (Hausser Scientific, cat. no. 0267110) - OMAX 40-2500X LED Digital Trinocular Compound Microscope USB Camera (OMAX, cat. No. M83EZ-SCP-C03S) - Fluorescence microscope (Nikon microscope Ti2 Eclipse or comparable) - 35 mm microscopy dishes, no. 1.5 Coverslip, 14 mm Glass Diameter, uncoated (Mattek) - 2/1 Holey Carbon-coated SiO 2 200-mesh Gold grids (Quantifoil Micro Tools GmbH, catalog number: Q2100CR1) - Tweezers (Dumont 3, 5) - Glass microscopy slides - Glass Petri dishes - Glow Discharge Cleaning System (PELCO easiGlow) - Leica EM GP2 plunger (Leica Microsystems) - Whatman filter paper grade 1 - Cryo grid boxes (custom-made) or (Thermo Fisher Scientific) - Autogrids with FIB cutout (MPI Martinsried or Thermo Fisher Scientific) - C-clips (Thermo Fisher Scientific) - C-clip insertion tools (Thermo Fisher Scientific) - EM Cryo CLEM system (Leica Microsystems) - Aquilos Cryo-Focused Ion Beam - Scanning Electron Microscope (Thermo Fisher Scientific) - Titan Krios G2 Cryo-Transmission Electron Microscope (Thermo Fisher Scientific), equipped with a BioQuantum-K3 (Gatan) REAGENT SETUP Enzyme solution: Prepare a solution containing 0.4 M mannitol, 20 mM MES (pH 5.7), 20 mM KCl, 1.5% (wt/vol) cellulase, and 0.3% (wt/vol) macerozyme.

Techniques: Electron Microscopy, Isolation, Fluorescence, Microscopy, Transgenic Assay