Chicken anti vimentin

In order to analyze the specificity of the adenovirus in vivo, the animals were injected with the adenovirus, and the brains were collected at 48 and 96 h. The rat brains were fixed in 4% PFA by immersion for 48 h. Free-floating frontal hypothalamic slices of 40 μm thickness were obtained by a cryostat and subsequently processed. Tissues were stained with chicken anti-vimentin (1:200; Millipore, Billerica, MA, USA), mouse anti-GFAP (1:200; Millipore), and rabbit anti-NeuN (1:5000; Abcam, Cambridge, MA, USA) antibody diluted in Tris-HCl buffer (pH 7.8) containing 1% bovine serum albumin. After extensive rinsing, the sections were incubated for 2 h at room temperature with Cy2- or Cy3-labeled secondary antibodies (1:200; Jackson ImmunoResearch Laboratories). TOPRO-3 (1:1000; Invitrogen) was used as nuclei staining. The slides were visualized and captured using confocal laser microscopy LSM 700 (Zeiss).

Cells from neurosphere differentiation cultures were fixed in 4% PFA with a 30% sucrose solution for 30 min at 37°C. For immunocytochemistry, cultures were preincubated for 1 h in blocking solution (10% goat serum, 0.1% Triton X-100, BSA), followed by overnight incubation with the appropriate primary antibody at 4°C. The following primary antibodies were used: mouse anti-hGFAP (1:500, Sternberger Monoclonal), chicken anti-vimentin (1:200, Millipore), rabbit anti-NG2 (1:200, Millipore), mouse anti-Olig2 (1:200, Millipore), chicken anti-Tuj1 (1:200, Millipore), rabbit anti-active caspase-3 (1:200, Abcam), rabbit anti-HSP27 (1:200, Abcam), and rabbit anti-cathepsin (1:200, Abcam). The corresponding secondary antibodies were incubated for 2 h (Alexa-Fluor 405, 488, 555, or 647 goat anti-mouse, chicken or rabbit; 1:500; Invitrogen), followed by incubation with DAPI (1:1,000, Sigma) for 10 min and rinsing before being mounted on glass slides with Fluorsave (Calbiochem). Analyses were performed with a Nikon 80i fluorescence microscope at 40× or 63× magnification.

Animals were injected with the adenovirus as described above, and brains were collected for immunohistochemistry 48 hr later. Rat brains were fixed for 24 hr in 4% paraformaldehyde (PFA), and thick frontal sections of the hypothalamus and brainstem (40 µm) and other brain regions were cut with a cryostat. Tissues were stained with chicken anti‐vimentin (1:200; Millipore, Billerica, MA, USA), rabbit anti‐NeuN (1:5,000; Abcam, Cambridge, MA, USA), mouse anti‐GFAP (1:200; Millipore), and mouse anti‐HuC (1:200; Invitrogen, Rockville, MD, USA). The antibody was diluted in Tris‐HCl buffer (pH 7.8) containing 8.4 mM Na₃PO₄, 3.5 mM KH₂PO₄, 120 mM NaCl, and 1% bovine serum albumin. Sections were incubated with the antibodies overnight at room temperature in a humid chamber. After washing, sections were incubated for 2 hr at room temperature with Cy2‐ (1:200) Cy3‐ (1:200) and Cy5‐ (1:200; Jackson ImmunoResearch Laboratories) labeled secondary antibodies. These samples were then counterstained with the DNA stain, HOECHST (1:1,000; Invitrogen). The slides were analyzed using confocal‐spectral laser microscopy (LSM 780 NLO, Zeiss).

Colonic tissue was harvested from female Pdgfra^H2BeGFP mice, fixed for 1 hour at room temperature in 4% paraformaldehyde in PBS (ChemCruz/Santa Cruz Biotechnology, USA), and incubated O/N in 30% sucrose in PBS at 4°C. Tissue was then kept in a 1:1 mix of 30% sucrose and optimal cutting temperature (OCT) (TissueTek/Biosystems Switzerland) for 30 minutes at 4°C, before embedding in OCT and being cooled to −80°C. OCT-embedded tissue was cryosectioned using a Microm HM560 Cryostat (Thermo Fisher Scientific, Switzerland) at 5 μm, dried for 2 hours at room temperature before either being directly used for immunohistochemistry, or stored at −80°C. Standard immunohistochemical protocols were performed with the following primary antibodies (1:100 dilution): mouse-anti-Acta2 (Sigma, Germany), goat-anti-Pdgfra (R&D Systems, USA), chicken-anti-Vimentin (Millipore, USA), and rabbit-anti-EpCAM (Abcam, UK). Secondary antibodies (1:400 dilution) were anti-rabbit, anti-mouse, anti-goat antibodies conjugated with Alexa Dyes (A555, A598, or A647) (Thermo Fisher Scientific, Switzerland). Sections were counterstained with DAPI, mounted with FluorSave reagent (Sigma), imaged on a Leica SP8 laser scanning confocal microscope (Leica, Switzerland), and subsequently processed using ImageJ (FIJI).