Dmi6000 inverted microscope

Before cryosectioning, forelimbs were embedded in Optimal Cutting Temperature compound (OCT, Sakura Finetek), frozen with dry ice-cooled isopentane and stored at -80°C until sectioned. 5 – 10μm cryosections were collected on His-bond⁺ glass slides (VWR) and washed with PBS for 3 times to remove any residual OCT before conducting AFM. Cryosections were processed following (Calve et al., 2010 (link)) and stained with primary antibodies against perlecan (1:50; Santa Cruz Biotechnology sc33707) and type VI collagen (1:200, Millipore AB7821) and then the secondary detection reagents [Alexa Fluor 647 anti-rabbit (1:500, Life Technologies), DyLight 550 anti-rat (1:250, Pierce), DAPI (1:500, Roche). Sections were imaged at 10x using a Leica DMI6000 inverted microscope and at 63x using a Zeiss 710 confocal microscope. Pictures were acquired using the same imaging parameters across the different genotypes and processed under identical conditions using Adobe Photoshop.