1 m nacl

Similar to Martinez-Haro et al. (2009), avian gizzard digestive juices were simulated by preparing a solution of 1 M NaCl (Sigma-Aldrich, St. Louis, MO) containing 10 g L^-1 of pepsin (Sigma-Aldrich, St. Louis, MO) and adjusting to pH 2.0 using hydrochloric acid (HCl; Fisher Scientific, Hampton, NH). The pH of the gizzard is typically more acidic (2.0–3.2) [55 ] than the intestines (5.2–7.2) [56 ,57 (link)], varying with the amount of food present in the GI tract [56 ]. Following previous studies [46 (link),55 ,57 (link),58 (link)], a pH of 2.0 was used to prepare the simulated gizzard solution.

dSTORM buffer consisted of a glucose oxidase/catalase-based oxygen scavenger system supplemented with a thiol in a buffer system. An enzyme stock solution (ES) was prepared at 0.1 kU/mL glucose oxidase (Sigma Aldrich), 1.2 kU/mL catalase (Sigma Aldrich), 4 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma Aldrich), 25 mM potassium chloride (KCl) (Acros Organics), 20 mM Tris-HCl pH 7.5, and 50% v/v glycerol (Riedel-de Haen) in ddH₂O. A glucose stock solution (GS) was prepared at 10% w/v glucose (Sigma Aldrich), and 10% glycerol in ddH₂O. ES and GS were stored in aliquots at −20 °C for up to 4 months. We prepared the imaging buffer by mixing 480 µL GS, 520 µL ddH₂O, 60 µL 1 M Tris-HCl pH 8, 60 µL 1 M NaCl (Sigma Aldrich), 12 µL 200 mM cyclooctatetraene (COT) (Sigma Aldrich) in dimethyl sulfoxide (DMSO) (Sigma Aldrich), 60 µL ES, and 12 µL 100% ß-mercaptoethanol (BME) (Sigma Aldrich) (preparation for measurement shown in Fig. 3b) or 8.4 µL BME (preparation for measurement shown in Figs 2d,e and 3c). Hence, the thiol concentrations in the imaging buffer were either 143 mM or 100 mM BME.

The extraction of polysaccharides from P. cruentum was performed using the method proposed by Abdala-Díaz et al. (2010) [26 (link)] with minor modifications. Once the culture of P. cruentum reached the early stationary phase, centrifugation was performed at 4500 rpm for 10 min, and the supernatant was recovered for polysaccharides extraction. The polysaccharides were extracted by selective precipitation with 2% Cetylpyridinium bromide hydrate (w/v) (Cetavlon) (Sigma-Aldrich Ref: 285315). The precipitated sulfated polysaccharides were dissolved with 4 M NaCl (Sigma-Aldrich), centrifuged at 5000 rpm for 10 min and then flocculated with 96% (v/v) ethanol (Sigma-Aldrich). The final pellet was recovered and introduced into a dialysis membrane (Cellulose dialysis tube, Sigma-Aldrich) for overnight dialysis at low osmotic pressure (1M NaCl) and agitation at 4 °C. After dialysis, the entire membrane content was recovered and washed with 96% EtOH. Final centrifugation (4500 rpm, 2 min, at room temperature) was applied to recover the polysaccharides. The polysaccharides were then frozen at −80 °C in absolute ethanol for 24 h and lyophilized (Cryodos Lyophilizer, Telstar). The sulfated polysaccharide-enriched extract will hereinafter be referred to as “PcSPs”.