Vectashield medium h 1400

The sections were treated with 0.1% Triton X-100 and PBS at room temperature for 15 min. After three washes with PBS, sections were incubated with 10% normal goat serum (ImmunoBioScience Corp., Mukilteo, WA, USA) in PBS at room temperature for 1 h. They were then washed thrice with PBS and incubated overnight at 4 °C in PBS containing biotinylated Wisteria floribunda agglutinin (WFA) (B-1355, Vector Laboratories; 1:200) and/or antibodies described in the subsequent text. After washing with PBS, the sections were incubated with Alexa Fluor 594-conjugated streptavidin (S11227; Molecular Probes, Eugene, OR) and/or the corresponding secondary antibodies (described in the subsection Antibodies and lectins) at room temperature for 2 h. The labeled sections were then rinsed with PBS and mounted on glass slides using Vectashield medium (H-1400; Vector Laboratories, Funakoshi Co., Tokyo, Japan). The prepared slides were stored at 4 °C until microscopic analysis was performed.

We treated the cryostat sections with 0.1 % Triton X-100 with PBS at room temperature for 15 min. After three washes with PBS, we incubated the sections with 10 % normal goat serum (ImmunoBioScience Corp., Mukilteo, WA) in PBS at room temperature for 1 h. Sections were then washed three times with PBS, and incubated overnight at 4 °C in PBS containing biotinylated WFA (B-1355, Vector Laboratories; 1:200) and/or the antibodies described in the subsection “Antibodies and lectins.” After washing with PBS, we incubated the sections with Alexa Fluor 594-conjugated streptavidin (S11227; Molecular Probes, Eugene, OR) and/or the corresponding secondary antibodies (described in the subsection “Antibodies and lectins”) at room temperature for 2 h. We rinsed the labeled sections again with PBS and mounted them on glass slides with Vectashield medium (H-1400; Vector Laboratories, Funakoshi Co., Tokyo, Japan). We stored the prepared slides at 4 °C until microscopy analysis was performed.

The sections were then treated with 0.1% Triton X-100 and PBS at room temperature for 15 min. After three washes with PBS, sections were incubated with 10% normal goat serum (ImmunoBioScience Corp., Mukilteo, WA, USA) in PBS at room temperature for 1 h. They were then washed thrice with PBS and incubated overnight at 4 °C in PBS containing biotinylated WFA (B-1355, Vector Laboratories; 1:200) and the antibodies described in the subsequent text. Subsequently, the sections were incubated with Alexa Fluor 594-conjugated streptavidin (S11227; Molecular Probes, Eugene, OR, USA) and the corresponding secondary antibodies (described in the Lectins and antibodies subsection) at room temperature for 2 h. The labeled sections were then rinsed with PBS and mounted on glass slides using a Vectashield medium (H-1400; Vector Laboratories, Funakoshi Co., Tokyo, Japan). The prepared slides were stored at 4 °C until further microscopic analyses.