Vs 120 slide scanner

After terminal anaesthesia by barbiturate overdose, mice were perfused transcardially with 4% paraformaldehyde and spinal cords processed for immunofluorescence as previously described^{60 (link),72 (link)}. Primary antibodies were: rat anti-Pecam1 (BD Biosciences 550274, 1:200). Secondary antibodies were: Alexa Fluor 555 Goat Anti Rat (1:200, Life Technologies, A21434). Immunofluorescence was imaged digitally on a slide scanner [Olympus VS-120 Slide scanner] or confocal microscope [Zeiss LSM880 + Airy fast module with ZEN 2 Black software (Zeiss, Oberkochen, Germany)]. Images were processed using ImageJ (NIH) or Imaris (Bitplane, version 9.0.0).

Prior to the beamtime, we mapped to identify the osteochondral interface using optical and fluorescence mosaic of the windows obtained using both Olympus VS120 Slide scanner and Zeiss LSM 710 Confocal Laser Scanning/Multi-photon Microscope with OlyVIA 2.9 software. Fluorescent mapping was undertaken at ~ 15 keV, with 5 mm × 5 mm areas mapped per sample at low resolution. We selected representative regions of interest with 1 mm × 1 mm size from these mappings. Next, we scanned these regions at high resolution and high sensitivity at parameters to achieve the best possible elemental maps. XFM photons were gathered at the Australian Synchrotron's XFM beamline as an event mode data stream^{55 (link)} using the Maia detector system^{56 (link)} and processed using the dynamic analysis method^{57 (link)} as implemented in GeoPIXE^{58 (link)}. The data were quantified using well-characterised metallic foils and exported as 32-bit tiffs with units in areal density (ng/cm²). The tortuosity index was described as the ratio of the meandering curve to the straight-line length between the endpoints^{59 (link)}.

Immunohistochemistry was performed as described previously^{28 (link)}. Perfused post-mortem tissue was cryoprotected in 30% sucrose in PBS for 48 h before being embedded in cryomatrix (Tissue Tek O.C.T, Sakura Finetek) and freezing. Ten- or 20-micrometre-thick transverse sections of the spinal cord were cut on a cryostat (Leica), immediately mounted on glass slides and dried. Sections were blocked with 10% bovine serum albumin in PBS for 60 min. Then sections were incubated with the following primary antibody diluted in blocking solution at room temperature overnight: rabbit anti-GFAP (1:500, Dako Z0334), cFos (1:2,000 Synaptic Systems 226003), vGluT1 (1:1,000, Synaptic Systems 135302). Slides were washed four times with PBS before the secondary antibodies (Alexa Fluor Conjugated, ThermoFisher Scientific, USA) were applied for 90 min in blocking solution; donkey anti-rabbit Alexa Fluor 647 (1:1,000, A-31573), donkey anti-rat Alexa Fluor 647 (1:1,000, A-48272), donkey anti-goat Alexa Fluor 647 (1:1,000, A-21447). Slides were washed four times with PBS and then cover slipped with Mowiol. Immunofluorescence was imaged digitally using a slide scanner (Olympus VS-120 Slide scanner) or confocal microscope (Zeiss LSM880 + Airy fast module with ZEN 2 Black software (Zeiss)). Images were digitally processed using ImageJ (ImageJ NIH) software or Imaris (Bitplane, v.9.0.0).