Mouse anti rfp

Mice were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA), brains were rapidly dissected out and placed into 4% PFA overnight. Brains were rinsed in PBS, cryoprotected, and sliced (30 μm). After blocking and permeabilization, slices were incubated in primary antibody (rabbit anti-tyrosine hydroxylase 1:500, Pel-Freeze; mouse anti-RFP 1:500, Rockland Immunochemicals; rabbit anti-cerebellin 1, 1:250, Abcam) overnight at 4°C. Slices were then incubated in secondary antibody (donkey anti-mouse Alexa568, donkey anti-rabbit Alexa647, 1:1000, Invitrogen) for 1 h at room temperature and mounted onto slides for imaging.

Barrel cortex sections were washed in TRIS buffer (TB) for 15 min, TRIS-buffered saline (TBS) for 15 min and TBS + 0.5% Triton X-100 (TBST) for 2×15 min, all at pH 7.6. Blocking was done for 90 min at room temperature in 0.25% bovine serum albumin/10% goat serum/TBST (Jackson Immuno Research). Sections were incubated for 48–72 h at 4°C with primary antibodies (i) chicken anti-GFP (Aves) diluted 1:1000, (ii) mouse anti-RFP (Rockland) diluted 1:1000, and (iii) rabbit anti-PV (Swant) diluted 1:5000 in blocking solution. After washing 4×15 min with TBST, secondary antibodies (i) Alexa Fluor 488-conjugated goat anti-chicken IgG, (ii) Alexa Fluor 568-conjugated goat anti-mouse IgG2a, and (iii) Alexa Fluor 633-conjugated goat anti-rabbit (Molecular Probes) were diluted 1:500 in TBST and sections were incubated for 4h at room temperature. 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).
For staining against oG, blocking solution was prepared with 3% bovine serum albumin/10% goat serum/TBST. Guinea pig anti-rabies glycoprotein antibody (kindly donated by A. Lüthi) was diluted 1:500 in blocking solution and combined with Alexa Fluor 633-conjugated goat anti-guinea pig IgG (Molecular Probes).