Rabbit anti tuj1

Adult mice were euthanized with CO2. Hairy skin from the top of each foot was removed and fixed in 100% acetone for 8 hours at room temperature, followed by immersion in 1mg/ml X-gal in staining solution (0.1 M Phosphate Buffer pH 7.5, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 20 mM Tris-HCL, 0.02% NP-40, 0.01% sodium deoxycholate) for 48 hours at 4°C. Tissues were then rinsed, post-fixed in 4% paraformaldehyde/1xPBS overnight, washed in PBS, submerged in 30% sucrose/1xPBS, and embedded in O.C.T. media. Sections of 15–20 μm thickness were cut and collected on glass slides and kept frozen until use. Antibodies on these sections included Rat anti-CD3 epsilon (1:100, NBP1-26685, Novus Bio) and Rabbit anti-C-Kit (1:100, D13A2, Cell Signaling Tech). For DRG neurons, whole ganglia were briefly fixed in 4% paraformaldehyde for 5 minutes, followed by X-gal staining as above for 1.5–2 hours, and embedded for cryosections as above. Sections (10 μm thickness) were counterstained with Rabbit anti-Tuj1 (1:1000, Sigma T2200) and DAPI. Brightfield and fluorescence images were acquired on a Zeiss Observer V1. Tuj1-positive neurons with nuclei in the plane of sectioning were counted manually, and percent overlay with X-gal staining were calculated.

Separate litters (Controls, N=6; food-restricted, N=5) were used for determining offspring hypothalamic protein expression (Western Blot) at two ages (1 and 21 days). Excess 1 day old male pups during litter culling were euthanized by decapitation and brains collected. At 21 days of age, one male offspring per litter was anesthetized by 5 % isoflurane/2 % oxygen by mask, subsequently euthanized by an overdose of pentobarbital (200 mg/kg i.p.) and brain collected. Hypothalami were obtained by dissecting semi-spheres adjunct to two sides of hypothalamus, the dorsal part removed, and the ventral part (~2 mm) used.
Western Blot was performed as previously reported by our group (Desai et al, 2008). Data were normalized to GAPDH (1:10,000, Chemicon). Primary antibodies used were DNMT1 (1:500, Santa Cruz), rabbit anti-Hes1 (1:1000, Santa Cruz), rabbit anti-Tuj1 (1:5000, Sigma) and rabbit anti-GFAP (1:10,000, DAKO).

Disassociated Control NPCs in differentiating medium were seeded in 24 well plates with pre-placed cover glasses (Fisher, catalogue number: 12-545-84 18CIR-1D). Following 5 day treatments with DNMT siRNA and 5-Aza, cells were fixed in 4% paraformaldehyde in PBS for 30 min and stained using rabbit anti-Tuj1 (1:500, Sigma), rabbit anti-GFAP (1:500, DAKO) and secondary antibody donkey anti-rabbit IgG-Alexa 488 (1:250). Secondary antibodies were donkey anti-rabbit IgG-Alexa 488 (1:250) or donkey antimouse IgG-Alexa 568 (1:250).