Rabbit anti vip

Pregnant females were euthanized by lethal intraperitoneal (i.p.) injection of pentobarbital (50 mg kg⁻¹), embryos were collected by caesarian cut and brains were dissected and fixed ON in cold 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer, pH 7.4. For postnatal brains, animals were deeply anaesthetized by i.p. injection of pentobarbital, transcardially perfused with 0.9% saline followed by cold 4% PFA and postfixed (ON) in cold 4% PFA. Brains were cut on a Vibratome (Leica, VT1000S) for IHC and for free-floating in situ hybridization. Sections were kept at 4 °C in 0.1 M phosphate buffer saline and were stained by IHC as described31 (link) with the following primary antibodies rabbit anti-GFP (1:500; Millipore), goat anti-GFP (1:1,000; Chemicon), mouse anti-human ovalbumin upstream promoter transcription factor 2 (COUP-TFII; 1:500; Perseus proteomics), goat anti-SP8 (1:50; Santa-Cruz), rabbit anti-NKX2.1 (1:100; Santa-Cruz), mouse anti-PV (1:1,000; Swant), rat anti- SST (1:500; Millipore), mouse anti-Reelin (1:500; Abcam), rabbit anti-VIP (1:500; Abcam), rabbit anti-NPY (1:1,000; Immunostar), rabbit anti-Calretinin (1:1,000; Swant). Secondary goat or donkey Alexa-488, -568 and -647 antibodies (Molecular Probes, Invitrogen) raised against the appropriate species were used at a dilution of 1:500–1,000 and sections were counterstained with Hoechst 33258 (1:10,000).

Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) at 85 °C for 20 min when required. IHC was performed as previously described^{27 (link)} with some changes. Briefly, sections were blocked for 90 min in a blocking buffer (2% Normal Donkey Serum (Gibco, Life Technologies Europe), 0.3% Triton X-100 (Sigma), 0.1% sodium azide (Sigma), 2% bovine serum albumin (Applichem, Baden-Dättwill, Switzerland)) and were incubated with a primary antibody in the blocking buffer for two nights at 4 °C. The primary antibodies used are the following: rat anti-CTIP2 (1:500; Abcam, Cambridge, UK), rabbit anti-CUX1 (1:250; Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-PV (1:1000; Swant), mouse anti-Reelin (1:500; Abcam), mouse anti-SATB2 (1:500; Abcam), rat anti-SST (1:1000; Chemicon, Millipore) and rabbit anti-VIP (1:500; Abcam). After rinsing, slices were incubated for 2 h at room temperature with the corresponding secondary donkey or goat Alexa-488, -568 and -647 antibodies (Molecular Probes, Invitrogen) raised against the appropriate species at a dilution of 1:1000 and were counterstained with Hoechst 33258 (1:10 000, Life Technologies).