Paraformaldehyde 4

All chemicals were prepared as concentrated solutions according to the recommendations of the different manufacturers. Compounds were aliquoted in Eppendorf tubes and used once, to avoid repeated freezing/thawing processes. Aliquots were stored at −80 °C for no longer than two months. Care was taken to protect photosensitive molecules from light by wrapping the test tubes in aluminum foil. Drugs were extemporaneously diluted at their respective final concentration in DMEM containing 10% FBS+ N2+ B27 minus Anti-Oxidant (AO). In order to study the impact of Glucose on Rotenone induced toxicity, two distinct DMEM formulations differing only in their glucose concentration were used. HG conditions correspond to DMEM, high glucose, Glutamax Supplement, Pyruvate (Thermofisher, Gibco, Cat ref 10569010) containing 4.5 g.L⁻¹ (25 mM) of glucose. LG condition corresponds to DMEM, low glucose, Glutamax Supplement, Pyruvate (Thermofisher, Gibco Cat ref 10567014) containing 1 g.L⁻¹ (5 mM) of glucose. Ten days after seeding, Rotenone (5 μM, Sigma-Aldrich) with or without mdivi-1 (20 μM, Tocris) or SP 600125 (10 μM) diluted in LG or HG DMEM were applied for 24 or 48 h according to the treatment. After treatment, neurons were fixed with Paraformaldehyde 4% (Sigma-Aldrich) for 10 minutes at room temperature then washed with PBS.

On day -2, 4T1-GFP cells were seeded in triplicate in a 6-well plate (3 × 10⁴ cells/well). On day -1, MIRB nanoparticles were added to the culture medium (25 μg/ml, overnight incubation). On day 0, culture medium was removed, cells were washed 3 times with PBS and replenished with full medium. Fluorescence microscopy was performed to confirm the intracellular localization of MIRB particles, and 7.5 × 10⁴ control J774 macrophages were added to MIRB-labeled 4T1-GFP cells. Cells were next imaged for 2 days using fluorescence microscopy (AxioVert S100 microscope, Zeiss). Exposure times were kept constant during experiments. After fluorescence imaging, cells were rinsed 3 times with PBS, fixed with paraformaldehyde 4% (Sigma-Aldrich, Bornem, Belgium), and washed 3 times with PBS containing 0.1% Triton X-100 (Sigma-Aldrich, Bornem, Belgium) for membrane permeabilization. Fixed cells were next incubated with a PBS solution containing 1% potassium ferrocyanide (Sigma-Aldrich, Bornem, Belgium)/1% HCl for 15 minutes. After Perls' Prussian blue staining of iron, cells were washed with PBS and brightfield pictures were taken (AxioVert S100 microscope, Zeiss).

Cells were fixed in paraformaldehyde 4% (Sigma Aldrich, Saint-Louis, MO, USA) and blocked in PBS 1x (Klinipath, Olen, Belgium) containing 0.3% TritonTM X-100 and 5% normal goat serum (Cell Signaling Technology). The cells were incubated overnight at 4 °C with antibody anti-ATP1A1 (M7-BP-E9, mouse, dilution 1:400) (ThermoFisher Scientific) and anti-Cav1 (3238, rabbit, dilution 1:200) (Cell Signaling Technology) diluted in PBS 1x, 0.3% TritonTM X-100 and 1% BSA (Sigma). The secondary antibodies were, respectively, Alexa FluorTM 555 Goat AntiMouse IgG and Alexa FluorTM 488 Goat Anti-Rabbit IgG (Invitrogen). Nucleus were stained using VectaShield-DAPI (Vector laboratories). Images were acquired using a confocal microscope (Nikon Ti2 A1RHD25, Tokyo, Japan).

Cytospin slides were fixed in paraformaldehyde 4% (Sigma-Aldrich). After 3 lavages in TBS, cells were incubated 10 min in TBS-0.3% Triton X-100, were washed 3 times in TBS, blocked in TBS-1% BSA-10% SVF during 45 min and incubated with primary anti-BAFF (Abcam, ab16081) or control isotype antibodies over night at 4°C. After 3 lavages in TBS, cells were incubated 1 h at room temperature with anti-rat IgM conjugated to Alexa 488. Cytospins were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, rinsed and coverslip were mounted with Mowiol (Sigma-Aldrich). For fluorescent intensity quantification, the gray value was calculated in each cell selected on untreated pictures. Cells were observed using a Nikon eclipse 80i microscope and images were treated using ImageJ software.

Grafts of 8 mm in diameter were isolated from OA tibial plateau and fixed in paraformaldehyde 4% (Sigma-Aldrich, Lyon, France), decalcified in EDTA 15% (Sigma-Aldrich, Lyon, France) for 3 weeks and embedded in paraffin. Sections (5 µm) were stained with HES (Merck Millipore, Fontenay sous Bois, France) and scored by two blinded observers according to the OARSI scales (grade 0: intact cartilage surface, normal matrix morphology and intact cells orientation; Grade 1: superficial fibrillation and cells death and hypertrophy; Grade 2: matrix depletion in superficial and middle zone and disorientation of chondrocytes columns; Grade 3: matrix vertical fissures into mid zone and formation of the chondrocytes clusters; Grade 4: loss of superficial and mid zone of cartilage, grade 5 and 6: no cartilage left and bone deformation is present) [34 (link)].

The spheroids were fixed with Paraformaldehyde 4% (Sigma-Aldrich, St. Louis, MO, USA) and embedded in paraffin. The paraffin sections were rehydrated by using both xylene and a descending-graded series of ethyl alcohol. After washing with the 50% alcohol, the sections were kept for five minutes in distilled water. Then, they were stained with haematoxylin (Bio-Optica, Milan, Italy) for 2 min, washed in distilled water, and then stained with Eosin (Bio-Optica) for 2 min; subsequently, they were washed in distilled water. Finally, they were dehydrated through an ascending graded series of ethyl alcohol and xylene and were mounted with Eukitt Solution (Orsatec GmbH, Kindler GmbH and Co., Bobingen, Germany).

On day 27 ± 2 of the differentiation the cells were detached and dissociated with Detachment Kit 2 (Promocell, Heidelberg, Germany) centrifuged 5 min at 200 g then fixed with paraformaldehyde (4%) for 10 min at RT (Sigma-Aldrich) and permeabilized with 90% cold methanol for 15 min at +4 °C . The cells were washed 3 times with PBS and then stained with Anti-Cardiac Troponin T-APC 1:100, recombinant human IgG1, clone REA400 (MiltenyiBiotec, Bergisch Gladbach, Germany) or CTL-I APC 1:100, Monoclonal REA Control (I) antibody human, clone REA293 (MiltenyiBiotec) diluted in PBS plus 0.1% Triton X-100 and 0.5% BSA for 45min at RT in the dark. The cells were washed and resuspended with PBS plus 0.5% BSA and collected on MACSQuant^® Analyzer 10 (MiltenyiBiotec) and analyzed using FlowJo.