Paraformaldehyde 4

To isolate PMN, heparinized blood was layered on an association of dextran and diatrizoate gradient with a density of 1.113 ± 0.001 (Granulosep: Eurobio, Les Ulis, France) following the manufacturer’s instructions. Cells were then distributed in a 24 well plate (2.10⁵ cells/well) on coverslips coated with L-lysine (Acros Organics, Belgium). PMN were then stimulated with serum from SSc patients or from controls (final dilution of 10%) for 4 h at 37 °C and 5% CO₂ in a final volume of 500 µL. Each condition was performed in duplicate. At the end of incubation, cells were washed and fixed by paraformaldehyde 4% (VWR, Darmstadt, Germany) as described by Brinkmann [20 (link)].

Cultured ITT fragments from three piglets were harvested for analyses on days 5, 10, 20, and 30 and then fixed in paraformaldehyde 4% (VWR, Belgium) overnight before being embedded in paraffin. Five-micrometer-thick sections on Superfrost^® Plus slides (VWR, Belgium) were used for histology, immunohistochemistry (IHC) and immunofluorescence (IF). Supernatants were retrieved every four to 5 days and stored at −80°C.

At the end of the study, animals were sacrificed by sodium pentobarbital overdose (180 mg·kg⁻¹) and their hearts were excised and snap frozen in isopentane cooled with liquid nitrogen. Contiguous 8 μm sections were obtained with a cryostat at − 22 °C for autohistoradiography and histological staining, respectively.
Distribution of ¹⁸F-FDG activity was recorded with an autohistoradiography system dedicated to the detection of electrons and positrons (µImager™, Biospace, France).22 (link) For Hematoxylin-Eosin-Safran (HES) staining, the sections were fixed in 95% ethanol, stained with hematoxylin for 1 minute, eosin and safran for 30 seconds each, before being dehydrated in ethanol 100% and xylene. For the Masson trichrome staining, the sections were fixed by immersion in Bouin solution for 15 minutes and picric acid for 5 minutes. The nuclei were stained with Weigert hematoxylin for 10 minutes, cytoplasm and smooth muscle with Biebrich solution and collagen fibers by immersion for 5 minutes in aniline blue.
For further immunohistology analyses, adjacent 5 µm sections were fixed with paraformaldehyde 4% (VWR, Fontenay-sous-Bois, France), incubated with a rabbit polyclonal antibody to determine macrophage infiltrates (anti-Vimentin antibody; 1:500; Dako, Les Ulis, France).