Spurr s resin

Vero cells were grown on Thermanox™ (Ted Pella, Redding, CA, USA) coverslips and infected SARS-CoV-2 and incubated for the stated times. Cells were rinsed with Hanks balanced salt solution, followed by fixation with 2% paraformaldehyde overnight at room temperature. Specimens were rinsed with PBS, permeabilized with 0.01% saponin in PBS for 5 min at room temperature and incubated with primary antibodies followed by peroxidase conjugated secondary antibody in 0.01% saponin in PBS. Samples were fixed for 1 h with 1.5% glutaraldehyde in 0.1 M sodium-cacodylate with 5% sucrose (pH 7.4), rinsed three times and developed using the Pierce diaminobenzidine (DAB) metal-enhanced substrate kit prior to embedding. Samples were embedded in Spurr’s resin (Ted Pella, Redding CA, USA) and micrographs were acquired using a 120 kV FEI BT Tecnai transmission electron microscope (Thermo Fisher/FEI, Hillsboro, OR, USA). Digital images were acquired with a Gatan Rio camera (Gatan, Pleasanton, CA, USA).

HeLa cells grown in T-25 mL flasks were infected with C. trachomatis L2 (Ct; MOI of 5) and treated with 5 μM iAkt from 2 h pi until its fixation at 24 h pi. Infected cell monolayers were fixed with 2% glutaraldehyde/PBS for 1 h at 37°C. Then, cells were removed with 1% gelatin/PBS, gently centrifuged (15 min at 1200 rpm) and washed three times with PBS. After that, cells were incubated with Osmium tetroxide/Potassium ferricyanide/PBS (1:1:1) for 90 min. Samples were dehydrated using increasing acetone series and embedded in Spurr’s resin (Ted Pella Inc., United States). Thin sections were cut with an ultramicrotome (Leica ultracut R, Austria) and stained with 1% uranyl acetate and Reynold’s lead citrate (Ted Pella Inc., United States) before they were observed with a Zeiss 900 electron microscope (Zeiss, Germany). Images were processed using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, United States).

Developing and mature fasicles were dissected and treated overnight with a fixative containing 4% (v/v) glutaraldehyde in 50 mM PIPES buffer (pH 7.2). Samples were post-fixed in aqueous 0.5% (w/v) OsO₄ for 2 h at 20 °C and then dehydrated in an ethanol series. The ethanol was then exchanged for propylene oxide, with subsequent infiltration of Spurr’s resin (Ted Pella, Redding, CA, USA). Thick sections (1 µm) were cut with glass knives using an Ultracut R microtome (Leica Microsystems, Buffalo Grove, IL, USA) and stained with toluidine blue. Sections were then photographed with a BH-2 Light Microscope (Olympus America Inc., Center Valley, PA, USA).

Selected samples (n = 2 per group) from each group were evaluated by SEM and TEM to characterize structural variations. For SEM, samples were coated with a 10–20 nm thick Au thin film using a sputter coater (Denton Vacuum Inc., Model # Desk II, Moorestown, NJ) and imaged using a Hitachi S-4300 field emission gun scanning electron microscope (Hitachi High Technologies America, Pleasanton, CA) at an accelerating voltage of 10 kV at working distances of <12 mm. For TEM, samples were further trimmed and embedded after ethyl alcohol and acetone dehydration in Spurr’s resin (Ted Pella, Redding, CA). Unstained, 70- to 80-nm-thick sections were examined by means of a JEM-1400 TEM (JEOL, Tokyo, Japan) at 120 kV following the general procedures described previously [16 (link)].