Sponges were grown on 35 mm glass bottom dishes (MatTek Life Sciences) and either infected with live sponge-derived algae or left untreated. Fixation and imaging were conducted as described in Hall et al. (2021) (link). Briefly, sponges were fixed in 4% paraformaldehyde and 1/4 Holtfreter’s Solution overnight at 4 °C. After washing and permeabilization, tissue was stained with Hoescht 33342 (1:200 dilution, Thermo Fisher Scientific, Waltham, MA) and Phalloidin Alexa 488 (1:40 dilution, Thermo Fisher Scientific, Waltham, MA) and imaged using an Olympus FV1200 laser scanning microscope using FluoView software.
Electron microscopy was performed as described in Hall et al. (2021) (link). Briefly, sponge samples infected with live algae were fixed in 2.5% glutaraldehyde, washed in 0.2 M cacodylate buffer (pH 7.4), and postfixed with 1% OsO4 and 1% uranyl acetate. Samples were dehydrated, infiltrated in propylene oxide, and embedded in Embed 812 plastic resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Micrographs were taken using a JEOL 1010 transmission electron microscope at the University of Richmond with an Advanced Microscopy Techniques XR-100 Digital CCD system.
Electron microscopy was performed as described in Hall et al. (2021) (link). Briefly, sponge samples infected with live algae were fixed in 2.5% glutaraldehyde, washed in 0.2 M cacodylate buffer (pH 7.4), and postfixed with 1% OsO4 and 1% uranyl acetate. Samples were dehydrated, infiltrated in propylene oxide, and embedded in Embed 812 plastic resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Micrographs were taken using a JEOL 1010 transmission electron microscope at the University of Richmond with an Advanced Microscopy Techniques XR-100 Digital CCD system.
