His bond glass slides

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.

To visualize the fibrillar structure of ECM within intact tissues, freshly harvested E14.5 forelimbs were embedded in Optimal Cutting Temperature compound (Sakura Finetek), frozen with dry ice-cooled isopentane and stored at 80°C. 17 µm-thick cryosections were collected on His-bond glass slides (VWR) and processed following (Calve et al., 2010 (link)) using the antibody concentrations defined in Table 2. Sections were imaged at 25× using a Zeiss LSM 880 confocal.