Fluorescence mounting medium

MPPs or LT-HSCs were sorted into the slide coated with poly-l-lysine (SIGMA). Cells were placed on ice for 30 min to permit cells to settle onto the slide and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then permeabilized with 0.2% TritonX-100 for 15 min at room temperature and blocked with 5% goat serum for 1 h at room temperature. Cells were incubated with the anti-p-H2A.X (Cell Signaling Technology, #9718, 1:200) or anti-Tom20 (Santa Cruz Biotechnology, sc-11415, 1:500) antibody overnight at 4 °C, washed and incubated in the Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, A11011, 1:1000) for 1 h at room temperature. Cell nuclei were stained with DAPI and mounted using fluorescence mounting medium (DAKO). Images were acquired with a Confocal Microscope A1R or super resolution microscope N-SIM (Nikon), and were processed with NIS-Elements software (Nikon). For quantitative analysis of mitochondrial morphology, images were first thresholded and then converted to binary images by using ImageJ software^{69 (link),70 (link)}. Individual particle (mitochondria) were analyzed for mitochondrial area and number of fragments.

After 24 h in culture, Myofibers were stained according to ref. ^{60 (link)}. Myofibers were washed with PBS twice and fixed with 4% PFA for 8 min at RT, followed by 2 times PBS wash. Permeabilization with 0.5% Triton X100 (T 8787, Sigma-Aldrich) for 8 min at RT, blocking with 20% Horseserum in PBS for 1 h at RT. Primary antibodies were diluted in PBS O/N at 4 C. Myofibers were washed twice with PBS + 0.05% Triton × 100. Secondary antibodies were diluted in PBS for 1 hr at RT, washed again twice with PBS + 0.05% Triton × 100 and mounted using Fluorescence Mounting Medium (S3023, Dako). More information on the antibodies used is provided in the Source Data file.

Brains were fixed as previously described. Next, 3 washes of 10 min each were performed using the Tris-Phosphate buffer. Subsequently, the slices were incubated with the primary antibody: Anti-Vimentin (1:400), Anti-Nestin (1:200), Anti-GFAP (1:200) and Anti-3F3-FMA (1:200), anti-MDA (1:100) throughout the overnight. Then, samples were washed 3 times for 10 min with Tris-phosphate buffer and incubated with the following secondary antibodies: Anti-Mouse Cy5, Anti-Chicken Cy3 and Anti-Rabbit cy2 (The Jackson Laboratory) for 2 h. Hoechst 33342 (1:1000) was used for nuclear staining. Finally, the sections were mounted using fluorescence mounting medium (Dako).

Standard immunological techniques were employed. Briefly, plated cells were rinsed and fixed with 4% paraformaldehyde prior to blocking for 1 hour with immunoblot. Primary antibody was applied overnight at 4°C, then after rinsing, goat anti-mouse Alexa-546 was applied. After a 4-hour incubation, the cells were rinsed, stained with DAPI (Thermo Fisher), and mounted with fluorescence mounting medium (DAKO). Cultures were imaged using a Nikon Eclipse TE2000-U microscope equipped with a SPOT RT Slider digital camera (Diagnostic Instruments). Primary antibody omission served as negative controls, and no immunoreactivity was observed.

SAA (30 μg.mL⁻¹) was incubated with pooled healthy PPP for 30 minutes at room temperature. Amytracker 480 and 680 were added to the samples and incubated at room temperature for a further 30 minutes. Clot formation was initiated with thrombin and the clots ( ± SAA) were air-dried for 4 minutes. This was followed by fixation with 10% neutral buffered formalin (NBF). After phosphate-buffered saline (PBS) (pH = 7.4) washing steps, samples were blocked with 5% goat serum (in PBS), and incubated with anti-human SAA antibody (1:500 in goat serum) (Anti-Serum Amyloid A antibody [EP11592-92]; ab190802) for one hour. After further PBS washing steps, the sample was incubated with secondary antibody (1:500 in PBS) (Goat Anti-Mouse IgG H&L Alexa Fluor® 488; ab150113) at room temperature in the dark for a further hour. The samples were finally washed and a coverslip was mounted with a drop of Dako fluorescence mounting medium on a microscopy slide for confocal analysis. The prepared samples were viewed on a confocal microscope (details above). SAA-antibody was viewed at an excitation of 488 nm, with emission measured with a GaAsP detector at 493–630 nm. Amytracker signal was viewed as above.