Alexa fluor 568 conjugated goat anti mouse igg2a

Barrel cortex sections were washed in TRIS buffer (TB) 1 × 15 min, TRIS-buffered saline (TBS) 1 × 15 min, and TBS + 0.5% Triton X-100 (TBST) 2 × 15 min, all at pH 7.6. For blocking, sections were incubated 90 min at room temperature in 0.25% bovine serum albumin/10% goat serum/TBST (Jackson ImmunoResearch). For primary antibody labeling, sections were incubated 48–72 h at 4 °C with (1) chicken anti-GFP (Aves; Cat#GFP-1020, RRID:AB_10000240) diluted 1:500 and (2) mouse anti-RFP (Rockland; Cat#200–301-379S, RRID:AB_2611064) diluted 1:2000. Sections were washed 4 × 15 min with TBST. For secondary antibody labeling, sections were incubated 4 h at room temperature with (1) Alexa Fluor 488-conjugated goat anti-chicken IgG (Molecular Probes; Cat#A11039) and (2) Alexa Fluor 568-conjugated goat anti-mouse IgG2a (Molecular Probes; Cat#A21134) diluted 1:500 in TBST. In some cases, sections were stained in addition with the primary antibody guinea pig anti-vGluT2 (Millipore; Cat#AB2251, RRID: AB_1587626) diluted 1:2000 and the secondary antibody Alexa Fluor 633-conjugated goat anti-guinea pig (Molecular probes; Cat#A21105) diluted 1:500. After washing 2 × 15 min with TBST and 1 × 15 min with TBS, sections were stained with DAPI, diluted 1:1000 in TBS. After several washes in TB, sections were mounted in Aqua-Poly-Mount (Polysciences).