Goat anti gfp antibody

Each animal was deeply anesthetized by intraperitoneal injection of pentobarbital (20 mg/kg), and then perfused with chilled phosphate-buffered saline, followed by 4% paraformaldehyde in 0.1 mol/l phosphate buffer. After post-fixation overnight, 50 µm thick sections were cut with a vibrating blade microtome (VT1000S; Leica, Wetzlar, Germany). For immunohistochemistry, the following primary antibodies were used: rabbit anti-GFP antibody (1:500, MBL, Nagoya, Japan), goat anti-GFP antibody (1:200, Abcam, Cambridge, UK), rabbit anti-Iba1 antibody (1:500, Wako, Osaka, Japan); rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Dako, Glostrup, Denmark); goat anti-PDGFRα antibody (1:100, R&D Systems, MN, U.S.A); rabbit anti-GST-π antibody (1:500, Enzo life sciences, NY, U.S.A.); rabbit anti-nestin antibody (1:200, Santa Cruz Biotechnology, CA, U.S.A.); goat anti-doublecortin (Dcx) antibody (1:100, Santa Cruz Biotechnology); mouse anti-betaIII tubulin (Tuj1) antibody (1:100, Santa Cruz Biotechnology); mouse anti-NeuN antibody (1:100, Millipore, MA, U.S.A.); mouse anti-Ascl1 antibody (1:100, BD Pharmingen, NJ, U.S.A.); rabbit anti-Sox2 antibody (1:100, Santa Cruz Biotechnology); mouse anti-NeuroD1 antibody (1:100, Abcam). Each primary antibody was detected by appropriate secondary antibodies conjugated with Alexa Fluor 488 or 555^TM (Molecular Probes, OR, U.S.A.).

Animals administered with tamoxifen for fate mapping were fixed by perfusion via the left ventricle with 3% PFA and 0.5% glutaraldehyde in PBS. After washing with PBS, segments of sciatic nerve and spinal cord were embedded with 4% low-melting-point agarose and sliced at 100 μm on a vibratome (Leica). Pre-embedding immunogold labeling was performed according to the manufacturer's protocol (Aurion). Briefly, following permeabilization and blocking, the tissue slices were incubated at 4°C with goat anti-GFP antibody (Abcam) for 48 h, followed by ultrasmall gold particle-conjugated anti-goat IgG (Aurion) for 48 h at 4°C. The samples were then subjected to a standard resin-embedding protocol incorporating a silver enhancement step after osmium tetroxide (0.5%) treatment. The ultrathin sections were examined on a Hitachi H600 transmission electron microscope.

The processing of the bladder samples was carried out at the A*STAR/IMCB histology facility (A*STAR, Singapore). Briefly, the bladder tissues were embedded in 2% agar for paraffin processing. For IFA, 4- to 5μm serial sections were cut longitudinally, deparaffinized in xylene (twice for 5 min at room temperature), rehydrated in 100% ethanol (twice for 2 min at room temperature), 95% ethanol (for 1minute), and 80% ethanol (for 1 minute). Antigen retrieval was performed by boiling slides for 30 min in 1mM EDTA buffer, pH 8. Slides were blocked in 1% FBS 0.4% TritonX-100 for 1 h at room temperature, and subsequently incubated overnight at 4°C with the following primary antibodies- goat anti GFP antibody (Abcam 1:500 dilution in blocking buffer) and rabbit anti Rab35 antibody (Novus biological, 1in 50 dilution in blocking buffer). For LAMP1 triple staining, the sections were additionally stained with rat anti LAMP1 antibody (abcam 1:200 blocking buffer). Sections were washed thrice with blocking buffer and were subsequently incubated with Alexa Fluor 488 (anti-rabbit), Alexa 594 (anti-goat) and Alexa 350 (anti-rat) conjugated secondary antibodies (1:500; Molecular Probes). The sections were then stained with DAPI and were analyzed by confocal microscopy.