A27040

Cells on a chamber slide were incubated with anti-O4 antibodies (1:200; #MAB345, Sigma-Aldrich) for 5 min and fixed in 4% paraformaldehyde solution for 15 min at 37 °C. Following permeabilization with PBS containing 0.2% Triton X-100 and 3% goat serum for 1 h, cells were incubated with Cy3-conjugated anti-GFAP (1:200; C-9205, Sigma-Aldrich) and anti-PU.1 antibodies (1:500; 2258S, Cell Signalling) at 4 °C. The next day, cells were stained using Alexa Fluor 488-conjugated anti-mouse IgM antibodies (1:500; A21042, Invitrogen), Alexa Fluor 647-conjugated anti-rabbit antibodies (1:500; A27040, Invitrogen) and Hoechst 33342 (1 mg/mL; Invitrogen). Percentage of cells positive for GFAP, O4 or IBA1 staining in astrocyte culture was quantified using a CellInsight CX7 High Content Screening Platform (ThermoFisher). All conditions were assessed in triplicate.

Cells were fixed in 4% paraformaldehyde (10 min), permeated with 0.2% Triton X-100 (15 min), and blocked with 1% bovine serum albumin (30 min). The anti-FGFR1 primary antibody (ab824, Abcam) was incubated for 90 min, and the AlexaFluor 647 goat anti-rabbit (A27040, Invitrogen) secondary antibody for 45 min. Then, cells were incubated with DAPI (Sigma) for 10 min to stain nuclei. A filter of 30-µm was used to remove cell aggregates. ImageStream^X Mark II Flow Cytometer (EMD Millipore) was used to acquire single cells images at 60× magnification. The acquired raw image file (.rif) contained among 500 and 2000 events (10–30 events per second). The analysis of single cells fluorescence intensity and nucleus diameter was performed by using IDEAS software (version 6.2.64.0). To consider only single cells, a dot plot showing area versus aspect ratio (AR) was created. To estimate FGFR1 intensity in nuclear region, we generated a morphology mask that defined nucleus stained by DAPI and to measure the fluorescent signal in nuclear area.

Anti-HER2 (ab214275, Abcam, Cambridge, UK), actin (A2066, Sigma-Aldrich, Oslo, Norway), goat-anti-rabbit-HRP (65–6120, ThermoFisher, Oslo, Norway), goat anti-mouse-AF488 (ab150117, Abcam, Cambridge, UK), goat anti-rat-PE (405406, Biolegend, Oslo, Norway), goat anti-rat-AF546 (A11081, ThermoFisher, Oslo, Norway), goat anti-rabbit-AF647 (A27040, ThermoFisher, Oslo, Norway), horse-anti-mouse-HRP (7076S, Cell Signaling Technology, Danvers, MA, USA) and Fixable Viability Dye eFluor™ 780 (eBioscience, Oslo, Norway).

To assess the fiber type expression of GSK3β, we performed immunofluorescent microscopy experiments on serial 10 μm cryosections obtained from O.C.T. (optimal cutting temperature) embedded soleus and EDL muscles from n = 3 mobile wild-type C57BL/6J male mice. One serial cryosection underwent immunofluorescent fiber type staining as previously described to demarcate the various fiber types with MHC I, IIa, IIx, and IIb isoforms.³⁰ (link)^,47 (link) The other serial cryosection underwent immunofluorescent staining with a primary antibody for GSK3β (9315, Cell Signaling, 1:2500 dilution) and an anti-rabbit Alexa Fluor 647 fluorescent secondary antibody (A27040, ThermoFisher Scientific, 1:5000 dilution). Once fiber types were identified with MHC isoform expression/fluorescence, GSK3β was quantified by converting the image to grayscale and quantifying the mean gray value with ImageJ. Within a single muscle, 10 of each fiber type were randomly analyzed.

For immunofluorescence staining, HUVECs were fixed using 4% paraformaldehyde (PFA) in HBSS+ for 10 min at room temperature. The fixative was aspirated, and the cells were rinsed once with HBSS+. Next, the cells were permeabilized for 2 min with 0.2% Triton X-100 in HBSS+ and washed once with HBSS+. The cells were blocked in 5% BSA in HBSS+ for 30 min and incubated with the primary antibody solution overnight at 4°C. Mouse anti-human CD144 (1:150; 555661, BD Biosciences, USA), sheep anti-human CD31 (1:150; AF806, R and D Systems, The Netherlands) and rabbit anti-human vWF (1:1000; A0082, Agilent Dako, USA) were used as the primary antibodies. The cells were washed with HBSS+, followed by an one-hour incubation with Hoechst (1:2000; H3569, Invitrogen, USA), rhodamine phalloidin (1:200; P1951, Sigma-Aldrich, The Netherlands) and the secondary antibody solution, containing Alexa Fluor 488-conjugated goat anti-mouse (1:250; R37120, ThermoFisher, USA), Alexa Fluor 488-conjugated donkey anti-sheep (1:250; A11015, ThermoFisher, USA) and Alexa Fluor 647-conjugated goat anti-rabbit (1:250; A27040, ThermoFisher, USA) antibodies. The cells were washed three times with HBSS+. High-quality Z-stack images of the stained cells were acquired using a high-content confocal microscope (Molecular Devices, ImageXpress Micro Confocal).