Rh pe

The fluorescent dye 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE, Avanti Polar lipids Inc., Birmingham, UK) was made up to 1 mg/mL in chloroform and used to label the phospholipids by adding 50 µL of the solution to 1 mL of cream. Emission was collected in the range 570–625 nm.
Wheat Germ Agglutinin Alexa Fluor 488 (WGA, Cergy Pontoise, France) was made up to 1 mg/mL in low salt TBS (20 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl₂, pH 7.2) and used to label and locate carbohydrate moieties. Fifty µL of solution was added to 1 mL of cream. Emission was collected in the range 490–550 nm. For samples single and dual stained with Rh-PE and WGA, samples were kept at room temperature in the dark for a minimum of 6 h before analysis. 0.5% (w/w) low melting point agarose (held at 45 °C until required) (Thermofisher Scientific, Waltham, MA USA) was added to all single and dual stained samples before imaging 10 µL of sample and 20 µL of the agarose were deposited onto slides and mixed gently before a coverslip was added. A 63x oil immersion objective was used to acquire images taken at 1024 × 1024 pixels.

Sulfo-DIBMA was obtained from Glycon Biochemicals (Luckenwalde, Germany). DMPC was a kind gift from Lipoid (Ludwigshafen, Germany). NBD-PE and Rh-PE were purchased from Avanti Polar Lipids (Alabaster, USA). Tris(hydroxymethyl)aminomethane (Tris), NaOH, NaCl, HCl, MgCl₂, CaCl₂, and CHCl₃ were purchased from Carl Roth (Karlsruhe, Germany) and Sigma–Aldrich (Steinheim, Germany). All chemicals were purchased in the highest purity available.