Aqueous mounting media

Mouse cecal segments were fixed in Carnoy’s fixative, embedded in paraffin, and serially sectioned to 5-μm sections. Section was stained with hematoxylin and eosin (H&E) for intestine architecture. Terminal mucin glycans were examined using a panel of FITC-conjugated lectins: Ulex europaeus agglutinin-1 (UEA-1) for terminal fucose; concanavalin A (CONA) for mannose, Dolichos biflorus agglutinin (DBA) for N-acetylgalactosamine, peanut agglutinin (PNA) for galactose, and wheat germ agglutinin (WGA) for N-acetylglucosamine (Vector Laboratories, Burlingame, CA) as previously described [33 (link)]. Briefly, de-paraffinized sections were incubated with citrate buffer pH 6 (Vector Labs) for 20 min in a pressure cooker and blocked with PBS containing 10% BSA. Sections were then stained in a humidified chamber with FITC-labeled lectin (10 μg/ml) for 1 h at room temperature. Sections were washed with PBS, counterstained with DAPI (Thermo Fisher, USA) for 5 min at room temperature, and mounted using aqueous mounting media (Sigma Aldrich). Sections were analyzed by confocal microscopy (Leica Dmi8), and fluorescence was semi-qualitatively calculated by tabulating mean pixel intensity using the ImageJ software (National Institutes of Health).

Slides were quenched of GFP signal first by heating at 90 °C for 10 min. After 5 min of defatting in xylene, slides were rehydrated in serial dilutions of ethanol (100%, 95%, 70%, 50%; 3 min each) and water (2 × 3 min). Then slides were incubated for 8 min in a filtered 1% aqueous solution of Thioflavin-S (Sigma) in the dark at room temperature. Washes with ethanol (70%, 2x3min; 95%, 3 min) and water (3 exchanges) followed before mounting with aqueous mounting media (Sigma).

After fixation, in vitro samples were gently washed 3 times with phosphate buffered saline (PBS, Thermo Scientific). Primary antibodies used for immunostaining included Anti-Histone H3 (citrulline R2 + R8 + R17, Abcam; Cat. #: ab5103). DNA was stained by the DNA dye Hoechst 33342 (Thermo Scientific; Cat. #: 62249). Secondary antibodies used included donkey anti-Rabbit IgG AlexaFluor 488 (Thermo Scientific; Cat. #: A-11008). After staining samples prepared with aqueous mounting media (Sigma-Aldrich) and cover-slipped for microscopy.