Immunofluorescent Staining of Spores

Spores were fixed in 85% methanol and incubated with rabbit-anti-spore antibodies^{47 (link)}, diluted 1:10.000 in PBS with 5% bovine serum albumin (BSA) and with 1:2000 diluted Alexa488 conjugated goat-anti-rabbit-IgG (Thermo Fisher Scientific, Whaltham, MA). Cellulose was stained with 20 µg/ml Calcofluor White (Sigma-Aldrich, St. Louis, MO). For whole mount staining, structures developed on polytetrafluoroethylene membrane (Merck Millipore, Billerica, MA), were fixed with 50% and 100% methanol, successively, and stained with 1:2000 diluted mouse-anti-GFP antibody and 1:2000 diluted Alexa-Fluor594 conjugated anti-mouse antibody (Thermo Fisher Scientific, Whaltham, MA). Structures were mounted in the presence of 3 µM DAPI and imaged using a Leica LP2 confocal microscope.