Leo 912

For histochemical analysis pieces (5–6 mm³) of skin and exorbital lacrimal gland from each animal (n = 8 for each experimental group) were embedded in paraffin. Serial paraffin sections (5 µm-thick) were stained with (1) 0.2% Toluidine blue in Walpole buffer at pH 4.2; (2) Alcian blue/safranin (AB/safranin) [18 (link)].
For electron microscopy, pieces of skin and exorbital lacrimal glands (3 mm³) were immersed in Karnovsky’s fixative in cacodylate buffer (pH 7.4) and then postfixed for 2 h in cacodylate buffer containing 1% osmium tetroxide [19 (link)]. Sections were examined using a Zeiss LEO-912 transmission electron microscope.

Cells have been seeded out in chamber slides and were incubated for 60 min and 24 h with a concentration of 1 mg/ml of each used nanoparticle. At the endpoints the cells have been washed with 0.1 M PBS buffer and fixed with yellow-fix mixture (2 % paraformaldehyde, 2 % glutaraldehyde, 0.02 % picric acid mixed with 0.1 M phosphate buffer). After this step fixation in 1 % osmium tetroxide in 0.1 M sodium cacodylate buffer took place. Dehydration steps, involving washing in rising concentrations of alcohol, followed consecutively. The final steps of sample preparation involved embedding in EPON resin, ultra-thin-cutting (80–100 nm), staining with uranyl acetate and lead citrate and investigation in a LEO912 (Carl Zeiss AG, Oberkochen, Germany) transmission electron microscope.

Electron microscopy was performed as previously described (Heilingloh et al., 2015 (link)). Briefly, in a first step, dialysis of H- and L-particle preparations against 20 mM HEPES was performed in a SnakeSkin pleated dialysis tube (10000-moleculare-weight cutoff, Thermo Scientific, Rockford, IL, United States) overnight at 4°C. Afterward, particles were seeded on carbon-coated 400-square-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA, United States) for 20 min at RT and fixed with 2% glutaraldehyde. Finally, particles were stained with 1% uranyl acetate (diluted in 50% ethanol) for 10 min and subsequently with lead citrate for 5 min. For data analysis a transmission electron microscope (Leo 912; Zeiss, Oberkochen, Germany) was used.

Eyes were collected and fixed in Karnovsky's fixative overnight at 4°C, washed in Ringer's saline and treated with 2% osmium tetroxide in 0.1 M potassium-sodium phosphate buffer (PSPB) for 4 h at room temperature. Eyes were rinsed three times with PSPB and the cornea and lens were carefully removed. The remaining eyecups were cut into four equal pieces under a dissecting microscope. Eyecups were dehydrated in a graded concentration series of ethanol (50-100%) and then in propylene oxide (PO). Infiltration of eyecups was performed in PO/Spurr's resin (2:1 vol:vol, 2:2 vol:vol, then 100% resin; overnight at 4°C for each). Each eyecup quarter was embedded in Spurr's resin and polymerized in a 70°C oven overnight. Sectioning was performed using an ultramicrotome with a diamond knife, and sections were collected and placed on mesh copper grids. Sections were then stained with uranyl acetate and lead citrate. TEM imaging was carried out using a LEO 912 (Zeiss) operating at 100 kV accelerating voltage. Sections from the peripheral retina were imaged and retinal cell types were identified by morphology, location and nuclear size (Jeon et al., 1998 (link)).

Vessels were liberated from dinosaur femora cortical bone using EDTA, pH 8.0 (see methods in references [1 (link),2 ,6 (link)]), then either fixed in a solution of 2% paraformaldehyde: 0.01% gluteraldehyde, or left unfixed. Vessels were then rinsed and embedded in 6% agar (Sigma). Samples were rinsed, then dehydrated in ethanol series from 50% to 100%. Some material was post-fixed in 1% OsO4 for 1 hour. Samples were infiltrated with acetone, followed by 1:1 acetone:Spurrs embedding medium, then 100% Spurrs to infiltrate under vacuum overnight. Sections were cut to 60–90 nm and placed on Au 200 mesh EM grids. Some sections were post-stained in uranyl acetate:lead citrate, others were not stained. Sections were imaged using a ZEISS LEO 912 with an acceleration voltage of 100 kV, coupled to a Proscan 2048–2048 digital CCD camera.