Orious 1100 w

Transmission electron microscopy (TEM) was used to evaluate the natural ability of SF to form β-sheet fibrils. SF hydrogel discs were contrasted through negative staining using 2% uranyl acetate for 5 seconds. TEM images were acquired using a JEOL JEM 1400 TEM (Tokyo, Japan) and digitally recorded using a CCD digital camera Orious 1100W Tokyo, Japan.

Campylobacter cocktail morphology immediately after inoculation and at time 60, 150, and 360 min in cooking salt were analyzed by transmission electron microscopy (TEM). Samples were fixed overnight with 2.5% glutaraldehyde/2% paraformaldehyde in cacodylate buffer 0.1 M (pH 7.4). Samples were washed in 0.1 M sodium cacodylate buffer and fixed in 2% osmium tetroxide in the 0.1 M sodium cacodylate buffer overnight, followed by new fixation in 1% uranyl acetate overnight. Dehydration was performed in gradient series of ethanol solutions and propylene oxide and included in Epon resin by immersion of samples in increasing series of propylene oxide to EPON (till 0:1 ratio) for 60 min each. Sample inclusion in EPON resin was performed in a silicon mould. Sections with 60 nm thickness were prepared on a RMC Ultramicrotome (PowerTome, USA) using a diamond knife and recovered to 200 mesh Formvar Ni-grids, followed by 2% uranyl acetate and saturated lead citrate solution. Visualization was performed at 80 kV in a (JEOL JEM 1400 microscope (Japan)) and digital images were acquired using a CCD digital camera Orious 1100 W (Tokyo, Japan), at the HEMS core facility at i3S, University of Porto, Portugal.

For transmission electron microscopy, 3 × 10⁵ CD14⁺ enriched cells were plated in an 8-well plate (Lab-Tek^® II CC2 TM) in 300 μL of medium. Macrophage differentiation, polarization and co-culture with iNKT cells were carried out as previously mentioned. Samples were fixed overnight with 2.5% glutaraldehyde/2% paraformaldehyde in cacodylate buffer 0.1 M (pH 7.4) and washed in 0.1 M sodium cacodylate buffer. Next, samples were fixed in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer overnight, followed by a new overnight fixation in 1% uranyl acetate. Dehydration was performed in gradient series of ethanol solutions and propylene oxide and included in EPON resin by immersion of samples in increasing series of propylene oxide to EPON (till 0:1 ratio) for 60 min each. Sample inclusion in EPON resin was performed in a silicon mold. Sections with 60 nm thickness were prepared on a RMC Ultramicrotome (PowerTome) using a diamond knife and recovered to 200 mesh Formvar Ni-grids, followed by 2% uranyl acetate and saturated lead citrate solution. Visualization was performed at 80 kV in a JEOL JEM 1400 microscope (Japan) and digital images were acquired using a CCD digital camera Orious 1100 W (Tokyo, Japan).