Anti calbindin

Mice were deeply anaesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Then, the brains were removed and postfixed in the same fixative for 2 h at 4 °C and subsequently cryoprotected in 30% sucrose in PB. Frozen sections, either 10 or 50 μm thick, were prepared on a microtome. Sections were washed with PBS and incubated for 1 h at room temperature in blocking buffer: 20% Block Ace (KAC Co., Ltd.), 5% normal goat serum (NGS), 0.1% Triton X-100, 0.1% azide in PBS. Then, the sections were incubated overnight with primary antibody in antibody dilution buffer (5% Block Ace, 5% NGS, 0.1% Triton X-100, 0.1% azide in PBS) at 4 °C. Sections were then washed with 0.1% Triton X-100 in PBS and incubated for 1 h with secondary antibody in antibody dilution buffer at room temperature. The antibodies used were as follows: anti-calbindin (1:500; Sigma-Aldrich), anti-CTCF (1:1000; Cell Signaling Technology), anti-VGluT2 (1:10,000; Millipore), anti-active caspase-3 (1:500; Cell Signaling Technology), anti-calnexin (1:500; Enzo), anti-KDEL (1:2000; MBL), and anti-IP3R (1:500; abcam). For haematoxylin and eosin (HE) staining, sections were stained with Mayer’s haematoxylin and eosin Y (Muto Pure Chemicals, Tokyo, Japan).

Cells were seeded on glass coverslips, fixed with 4% PFA (Sigma-Aldrich) and incubated overnight at 4°C with the following primary antibodies: anti-PanCytokeratin (Mouse 1:200, Dako, Glostrup, Denmark), anti-Epcam (Mouse 1:1000, clone HEA-125, GeneTex, Irvine, CA, USA), anti-AQ-1 (Mouse 1:50, clone B-11, Santa Cruz Biotechnology, Heidelberg, Germany), anti-CD13-PE (Mouse 1:25, Biolegend, San Diego, CA, USA), anti-CD13-FITC (Mouse 1:25, Abcam, Cambridge, UK), anti-N-cadherin (Rabbit 1:50, Abcam, and Mouse 1:50, clone 32/N, Becton Dickinson, San Josè, CA, USA), anti-Calbindin (Mouse 1:100, clone CB-955, Sigma-Aldrich), anti-E-cadherin (Mouse 1:50, Becton Dickinson, and Rabbit 1:50, Cell Signalling Technology, Danvers, MA, USA) and anti-Paxillin (Mouse 1:50, Becton Dickinson). When necessary, the secondary antibodies Alexa 488 conjugated anti-mouse IgG and Alexa 594 conjugated anti-rabbit IgG (1:100, Molecular Probes, Carlsberg, CA, USA) were used. Stress fibers were labeled by Alexa-Fluor-594-phalloidin (1:100, Molecular Probes) and nuclei counterstained with Mounting DAPI (Molecular Probes). Immunofluorescence images were obtained with a Zeiss LSM810 confocal microscope, using a 63x objective, equipped with Zen2009 software (Zeiss, Oberkochen, Germany).

Primary antibodies targeting the following proteins were used for western blot and tissue sections: FGF9 (Abcam, EPR19937), Olig1 (Millipore, MAB5540), NeuN (Millipore, ABN78), Iba-1 (Abcam, ab5076), anti-GFAP (Millipore, MAB360), anti-Calbindin (Sigma, C2724), Caspase-3 (Santa Cruz, sc-373730), GABA (Sigma, A2052), VGAT (SYSY, 131011), Calbindin (Sigma, C2724), GAPDH (Abcam,ab128915), Adcy5/6 (Santa Cruz, sc-514785), p-ERK (Cell Signaling, #4370), ERK (Cell Signaling, #4695), p-Akt (Immunoway, YP006), Akt (Immunoway, YT0176), and β-actin (Proteintech, 60008-1-Ig). Secondary antibodies for western blot were from Rockland Immunochemicals, USA, and fluorescein isothiocyanate secondary antibodies were from Jackson ImmunoResearch, West Grove, PA. Hoechst stain (1:100, Invitrogen), RNeasy Lipid Tissue Mini kit (QIAGEN, 74804), SQ22536, bupivacaine-HCl, pentylenetetrazol, GABA, and Glu were purchased from Sigma (Madrid, Spain).

Mice were fixed by cardiac perfusion with 0.1 M phosphate buffer containing 4% paraformaldehyde and 4% sucrose for light microscopy, or with 0.1 M phosphate buffer containing 1.5% paraformaldehyde and 3% glutaraldehyde for EM. Brain tissues were excised and processed for morphological analysis. For light microscopic analysis, 10-µm cryosections were cut and immunolabelled with anti-GFAP (Sigma) and anti-calbindin (Sigma) antibodies.