To identify elastic microfibrils, elastin staining was performed using a Verhoeff van Gieson Elastic Stain Kit (Cat# HT25A, Sigma) in whole eye cross-sections according to the manufacturer’s protocol. Images were captured and analyzed (KEYENCE microscope).
For two-photon imaging, ex vivo mouse eyes were labeled intact, without dissection or sectioning, with Sulforhodamine-B (Thermo Fisher Scientific), a water soluble stain of elastic microfibrils. Microscopy was performed on a Leica SP5 microscope (Leica Microsystems, Heidelberg, Germany) coupled to a Chameleon Ultra-II multiphoton laser (Coherent, Santa Clara, CA) through inverted 20X/0.7NA or 63X/1.3NA objectives. We used 850 nm excitation, pulsed, and focused through green (525/50 nm) or red (585/40 nm) filters (Chroma, Bellows Falls, VT) onto a non-descanned photomultiplier tube detector (Hamamatsu, Bridgewater, NJ). Images were collected as multiple channel z-stacks using 512 × 512 or 1024 × 1024 pixel frames, 16-bit grayscale resolution, and 16X line averaging using 1–8 μm step sizes. Images were viewed and processed on LAS AF and AF Lite 2.2.1 (Leica), Image J (NIH; Bethesda, MD), Photoshop CS5 (Adobe, San Jose, CA) and Imaris (Oxford Instruments). Methods reported here have previously been described101 (link), 106 (link)–108 (link).
For two-photon imaging, ex vivo mouse eyes were labeled intact, without dissection or sectioning, with Sulforhodamine-B (Thermo Fisher Scientific), a water soluble stain of elastic microfibrils. Microscopy was performed on a Leica SP5 microscope (Leica Microsystems, Heidelberg, Germany) coupled to a Chameleon Ultra-II multiphoton laser (Coherent, Santa Clara, CA) through inverted 20X/0.7NA or 63X/1.3NA objectives. We used 850 nm excitation, pulsed, and focused through green (525/50 nm) or red (585/40 nm) filters (Chroma, Bellows Falls, VT) onto a non-descanned photomultiplier tube detector (Hamamatsu, Bridgewater, NJ). Images were collected as multiple channel z-stacks using 512 × 512 or 1024 × 1024 pixel frames, 16-bit grayscale resolution, and 16X line averaging using 1–8 μm step sizes. Images were viewed and processed on LAS AF and AF Lite 2.2.1 (Leica), Image J (NIH; Bethesda, MD), Photoshop CS5 (Adobe, San Jose, CA) and Imaris (Oxford Instruments). Methods reported here have previously been described101 (link), 106 (link)–108 (link).
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