Embed 812 embedding kit

Materials were acquired from the following sources: Gold chloride, poly (ethylene glycol) (PEG), poly-D-lysine (PDL), bovine serum albumin (BSA), colchicine and paraformaldehyde (PFA): Sigma-Aldrich, USA; Trisodium citrate dihydrate, papain, nitric acid, hydrogen peroxide, hydrochloric acid, TRIS-buffer, triton X-100, di-sodium hydrogen phosphate and potassium dihydrogen phosphate: Merck Chemicals, USA; Collagenase-II, dispase-II, fetal bovine serum (FBS), penicillin–streptomycin mix and trypsin–EDTA: Gibco, USA; Hank's buffered saline solution (HBSS), Ham’s F-12 medium and Dulbecco’s modified Eagle’s medium (DMEM): Lonza Chemicals, USA; Isolectin B4, DyLight 594-IB4, FITC-IB4, DyLight 488 horse anti-mouse IgG antibody and Vectashield hardset mounting medium: Vector Laboratories, USA; Glutaraldehyde (EM grade), osmium tetroxide (OsO₄), sodium cacodylate trihydrate and EMBed-812 embedding kit: Electron Microscopy Sciences, USA; Hoechst 33342: Santa Cruz Biotechnology, Inc. USA; Anti β-III tubulin monoclonal antibody: Cell Signaling Technology, Inc USA; Anti PGP9.5 antibody: Abcam Plc UK; Microfluidic devices (SND 150): Xona Microfluidics, LLC, USA.

Gravid adults were frozen substituted in acetone (J.T. Baker, Phillipsburg, NJ, USA) containing 2% osmium tetroxide (Electron Microscopy Science, Hatfield, PA, USA) and 0.2% uranyl acetate (Electron Microscopy Science) in a Leica EM automatic freeze substitution machine (Leica). The temperature was raised from −90 °C to room temperature for 22 h (5 °C/h). The samples were dehydrated in 100% ethanol (Sigma-Aldrich) for 24 h, followed by two washes. The washed samples were embedded in the EMBed-812 embedding kit (Electron Microscopy Science) and incubated at 70 °C for 48 h. The 70 nm thin sections were cut by using a Reichert ultracut S microtome (Leica). Thin sections were collected on Formvar-coated copper grids (Electron Microscopy Science) and post-stained with 0.8% uranyl acetate (Electron Microscopy Science) in 40% methanol (J.T.Baker) followed by aqueous lead citrate (Electron Microscopy Science). Thin sections were viewed in a JEM-1230 transmission electron microscope (JEOL, Akishima, Tokyo, Japan).

About 1 mm³ hippocampus tissues were dissected from P8 mice (after three times of treatment) and fixed in 2.0% glutaraldehyde for 2 hrs at 4°C. The tissues were rinsed in 0.1 M cacodylate buffer and postfixed in 1% OsO₄ for 2 hrs and then dehydrated by a series of graded ethanol. Then infiltration was done by first 1 : 1 resin : acetone for 1 hr, 2 : 1 resin : acetone for 1 hr, pure resin for 1 hr, and finally pure resin overnight. After that, the hippocampus tissues were embedded in a 60°C oven for 48 hrs. 70% ethanol was saturated with uranylacetate to enhance the contrast.
The hippocampus tissues were embedded in EMBED-812 EMBEDDING KIT (Electron Microscopy Sciences). Ultrathin sections (70–80 nm) were cut by Ultracut ultramicrotome (Leica UC6, Germany), mounted on pioloform-coated 50 mesh grids, and contrasted with 0.5% lead citrate for 5 mins. Ultrastructural changes of hippocampus synapses were observed and examined under a transmission electron microscope (PHLIP-CM-120, Holland). The total number of mice for transmission electron microscopy was twelve and the number of each group was three.

Immune cells were incubated in a 15 mL tube (Thermo Scientific, USA) (1 μg mL⁻¹ final concentration for 24 h) with gentle agitation, followed by fixing as described above. After intensive washes, cells were post-fixed overnight at 4°C with 1% osmium tetroxide in cacodylate buffer. Samples were washed three times in cacodylate buffer at 4°C and double distilled H₂O, warmed to the RT and embedded into 4% low-melt agarose. Solidified agarose was cut into 1 × 1 mm cubes, dehydrated in ethanol series (25, 50, 75, 90, 96, and 100%), and embedded into the epoxy resin (EMBed-812 Embedding kit; Electron Microscopy Sciences). Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a FEI Morgagni 268(D) electron microscope (FEI, Czech Republic).

CD14-positive cells were treated with 22.5 pM CyaA toxin or CyaA-AC⁻ toxoid for 5 days as described above and fixed for 2 h with 2% glutaraldehyde–PBS. After three washes with ice-cold PBS, cells were postfixed with 0.5% osmium tetroxide–PBS and incubated overnight at 4°C. Fixed and washed samples were dehydrated with ethanol using a standard procedure. Cells were embedded in epoxy resin (EMBed-812 embedding kit; Electron Microscopy Sciences). Ultrathin sections were visually contrasted using lead citrate and uranyl acetate (54 (link)). Final samples were examined using an FEI Morgagni 268(D) electron microscope (FEI, Brno, Czech Republic) at 80 kV. Digital images were recorded with a MegaViewIII slow-scan camera and processed by AnalySis 3.2 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany) using standard software modules (shading correction, digital contrast enhancement).

Fixed nerves were then processed by the Electron Microscopy platform at the Institute of Neurosciences in Montpellier (INM, MRI-COMET). Samples were post-fixed with osmium 0.5% and K4FeCN6 0.8% 24 h at room temperature in the dark, went through dehydration in successive ethanol baths and embedding in Epoxy resin (EMbed-812 Embedding Kit, Electron Microscopy Sciences, France) using a Leica AMW machine. They were finally incubated 36 h in an oven at 60 °C. Nerves were cut with Leica Reichert Ultracut S ultramicrotome into semi-thin sections of 700 nm—1 µm. Sections were stained with Toluidine Blue and imaged using an AxioScan slide scanner (Zeiss, France). The total number of myelinated axons, the mean axon diameter (100 axons per nerve randomly distributed throughout the entire section) and the g-ratio (axon diameter over the full fiber diameter) were measured using ImageJ software and GRatio plugin.

Immediately upon surgical extraction, the sural nerve and buccal nerve branch specimens were placed in a buffered fixative containing formaldehyde and glutaraldehyde for 24 hours (Trump’s Fixative, Electron Microscopy Sciences, PA, USA). Nerve specimens were then post-fixed with 2% osmium tetroxide diluted in 0.1 mol/L cacodylate buffer overnight before being embedded in epoxy resin (EMBed-812 Embedding Kit, Electron Microscopy Sciences, PA, USA). The resin embedded specimens were then cut at 2 μm thickness using an ultramicrotome and stained with 1% toluidine blue. Images of the semithin sections were then captured under a light microscope. Numbers of myelinated fibers and fascicles were manually counted using ImageJ.