Lx112 resin

Tissues were excised, washed in PBS, cut along the mesenteric plane, pinned flat, and then fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield PA) and 2.5% formaldehyde (Electron Microscopy Sciences), in 0.1M cacodylate buffer pH 7.4 containing 0.1 mM EGTA for 10 minutes at RT with gentle flushing. The tissue was then cut into small pieces, and fixed for an additional 1 hour in the same fixative at RT. Tissues were washed with 0.1 M cacodylate buffer, and then loaded into a planchette (Technotrade International, Manchester, NH) with PBS containing 20% BSA and 5% FBS, and subjected to high pressure freezing using a Wohlwend High Pressure Freezer (Technotrade International). Rapid freeze substitution, as described [79 (link)], was done using 1% osmium tetroxide, 0.5 % uranyl acetate, 95% acetone and 5% dH2O. After freeze substitution, the tissue was infiltrated with graded acetone into LX112 resin (Ted Pella, Inc. Redding ,CA). Ultrathin sections were cut with a Leica Ultracut E ultramicrotome (Leica Microsystems, Wetzlar Germany), placed on formvar and carbon coated grids, and then stained with 2% uranyl acetate (Electron Microscopy Sciences) and lead citrate (Sigma-Aldrich). Grids from each treatment were imaged using a JEOL 1400 electron microscope (JEOL USA, Peabody, MA) equipped with an Orius SC1000 digital CCD camera (Gatan, Pleasanton, CA).

Coculture experiments took place in 24 well plates at MOI 100. At T0 h, T24 h, and T48 h, samples were fixed in 3.5% glutaraldehyde for 45 min at 4 °C in NSS and post-fixed with 1% osmium tetroxide (OsO₄) in 0.1 M sodium Sorenson’s buffer for 1 h. Dehydration in increasing ethanol concentration solutions was followed by direct embedding of the samples from the surface in LX112 resin (Ted Pella, Redding, CA, USA). Ultrathin sections were stained with uranyl acetate followed or not by lead citrate at the CIQLE (Centre d’Imagerie Quantitative Lyon Est, platform of UCBL Lyon1 University) or at the PiCSL-FBI core facility (IBDM UMR CNRS 7288, Aix-Marseille University) and examined with a Tecnai G2 electron microscope (FEI, Hillsboro, OR, USA) operating at 200 kV at the PiCSL-FBI core facility.