Rabbit anti tet1

After euthanization, the dorsal quadrants of the lumbar enlargement (L4-5) of animals were dissected and collected for further study. The dissected dorsal horn (L4-5) samples were homogenized in a Tris-HCl solution (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS with a complete protease inhibitor mixture) (Roche, Upper Bavaria, Germany). The homogenates were solubilised 1 hour at 4 °C, and centrifugated at 4 °C for 20 min at 18,700 g. A bicinchoninic acid assay (BCA) was used to determine the protein concentration. The proteins were separated on an acrylamide gel and transferred to a polyvinylidene difluoride membrane and then incubated in either rabbit anti-Tet1 (1:500, Millipore, Billerica, Massachusetts), rabbit anti-BDNF (1:4000, Abcam, Cambridge, UK), or mouse anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:4000, Santa Cruz Biotechnology, Santa Cruz, CA) for one hour at room temperature. The blot was then washed and incubated in either peroxidase-conjugated goat anti-rabbit IgG (1:8000, Jackson ImmunoResearch, West Grove, PA) or goat anti-mouse IgG (1:8000, Jackson ImmunoResearch, West Grove, PA) for one hour at room temperature. The protein bands were visualized using an enhanced chemiluminescence detection kit (ECL Plus, Millipore, Billerica, Massachusetts) and then analyzed using a densitometry (Science Lab 2003; Fuji, Tokyo, Japan).