Dapi 4 6 diamidino 2 phenylindole

Rat peritoneal tissue sections were prepared as aforementioned. In brief, after treated with primary antibodies (Table S1) for 2 h at room temperature, sections were washed and allowed to react with secondary antibody goat anti-mouse IgG conjugated with Alexa 546 or donkey anti-goat conjugated with Alexa 488 (Invitrogen, Carlsbad, CA) or goat anti-rabbit IgG conjugated with Cy3 (Sigma-Aldrich) for 45 min at room temperature. The slides were visualized with an anti-fading reagent containing DAPI (4′,6-diamidino-2-phenylindole) (Nakarai Tesque, Kyoto, Japan) for nuclear staining. They were digitalized using the Panoramic MIDI digital slide scanner at its highest resolution.

All procedures were performed according to the method described elsewhere (55 ). In brief, algal samples were fixed in 4% paraformaldehyde (Wako, Osaka Japan), spotted on an aminosilane-coated glass slide (Matsunami Glass, Osaka, Japan), and air-dried, followed by successive dehydration in 50%, 80%, and 100% ethanol. The samples were hybridized with a CF319a probe (5ʹ-TGGTCCGTGTCTCAGTAC-3ʹ) labeled with the fluorophore FAM (Eurofins Genomics) in a hybridization buffer (35% formamide, 0.9 M NaCl, 20 mM Tris-HCl, and 0.01% SDS) at 46°C for 2 h. After being washed with a washing buffer (0.08 M NaCl, 20 mM Tris-HCl, 5 mM EDTA, and 0.01% SDS), the samples were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Nacalai Tesque, Kyoto, Japan) in Vectashield mounting medium (Vector Laboratories, CA, USA) and observed under a confocal microscope (LSM 700; Zeiss, Oberkochen, Germany).