Alexa fluor 488 conjugated donkey anti sheep igg

Cells plated on gelatin-coated glass slides (Nunc Lab-Tek II Chamber Slide System) were allowed to attach overnight. Cells were fixed for 20 minutes in 4% Paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA, USA), then incubated in blocking solution consisting of 2% normal donkey serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween 20, 0.05% sodium azide in 1× PBS (w/o Ca2+/Mg2+), pH 7.2 for 30 minutes, followed by 30-minute blocking in Protein Block, Serum-free (Dako, Carpinteria, CA, USA) + 0.1% Triton X-100. Sheep anti-Rab27a (Thermo Fisher) or an equivalent concentration of sheep IgG (Thermo Fisher) was diluted 1:40 in donkey serum blocking solution for 1 hour at room temperature, followed by Alexa Fluor 488-conjugated donkey anti-sheep IgG (Thermo Fisher). Cells were mounted in ProLong Gold Antifade with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher) and visualized on an inverted Nikon A1R fluorescent microscope. Identical imaging parameters (objective, light intensity, gain, exposure) were used between slides and samples.

The following lectins and primary antibodies were used for staining: biotinylated WFA (B-1355, Vector Laboratories; 1:200), mouse anti-PV (clone PARV-19, P3088; Sigma-Aldrich Japan, Tokyo, Japan; 1:1000), mouse anti-aggrecan (Cat-315; MAB1581, MERCK; 1:1000), rabbit anti-aggrecan (AB1031, Millipore, Tokyo, Japan; 1:200), goat anti-tenascin-R (AF3865, R&D Systems, Minneapolis, MN, USA; 1:200), sheep anti-brevican (AF4009, R&D Systems; 1:200), and mouse anti-glutamate decarboxylase (GAD67) (clone 1G10.2, MAB5406; Millipore, Bedford, MA; 1:1000).
The following secondary antibodies were used for visualization: Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin (Ig)G (ab150113; Abcam, Cambridge, MA; 1:1000), FITC-conjugated anti-mouse IgM (sc-2082, Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000), Texas Red-conjugated goat anti-rabbit (TI-1000; Vector Laboratories, Funakoshi Co., Tokyo, Japan), DyLight488-conjugated horse anti-goat IgG (DI-3088; Vector Laboratories; 1:500), Alexa Fluor 488-conjugated donkey anti-sheep IgG (A-11015; Thermo Fisher Scientific, Kanagawa, Japan; 1:1000), and streptavidin-conjugated Alexa Fluor 594 (S11227, Thermo Fisher Scientific; 1:1000).