Rabbit anti rest

Cells were cross-linked with 1% paraformaldehyde for 15 mins and Chromatins were sheared into an average 200–400 bp in length by sonication (Diagenode) and immunoprecipitated with following antibodies: rabbit anti-REST (Abcam), rabbit anti-CoREST (Upstate), rabbit anti-SIN3A, rabbit anti-HDAC1 (both from Proteintech). Immunoprecipitated DNA fragments were collected by magnetic beads (Active Motif), purified, and subjected to real-time PCR using primers specific to regions spanning predicted REST binding site on Nppa and Nppb. The forward and reverse primers for NRSE_b are respectively 5′-GCCTGGAAATCGGTTGAGGA-3′ and 5′-TTCCCAAACAACACCCACCA-3′. The forward and reverse primers for NRSE_a are respectively 5′-TCTAGTGGGGTCTTGCCTCT-3′ and 5′-GTCTGTCCTTGGTGCTGAAGT-3′. Data were normalized to values of the input DNA. REST binding site were identified using the Jaspar database (http://jaspar.genereg.net/).

All reagents were from Sigma unless otherwise stated. Standard protocols were used as previously described57 (link)58 (link). Mouse anti-β-actin, 1:10000, Sigma, A5441; Mouse anti-HIF-1α,1:1000, BD Pharmingen, 610958; Rabbit anti-REST, 1:1000, Abcam, ab28018; Rabbit anti-Lamin A/C, 1:1000, Cell Signalling, 2032; Mouse anti-α-Tubulin, 1:2000, SCB, sc-8035.

Free-floating basal forebrain in vivo (every 6^th section; approximately Bregma: 1.00 mm – 0.20 mm based on the atlas of Paxinos and Watson [26 ]) and ex vivo immunohistochemistry experiments were conducted as previously described [13 (link)–15 (link), 32 ]. Sections were incubated in a primary antibody solution containing blocking solution with either goat anti-ChAT (Millipore, Temecula, CA, USA, Cat. #AB144P), rabbit anti-parvalbumin (Abcam, Cambridge, MA, USA, Cat. #ab11427), mouse anti-NeuN (Millipore, Cat. #MAB377), or rabbit anti-REST (Abcam, Cat. #ab202962). Negative control for non-specific binding was conducted on in vivo sections employing the above-mentioned procedures, omitting the primary antibody.
Immunohistochemical assessment of paraffin-embedded post-mortem human basal forebrain sections were conducted as previously described [12 (link), 32 , 33 (link)]. Slides were incubated in a primary antibody solution containing Dako antibody diluent (Dako North America, Carpinteria, CA, Cat. #S0809) and rabbit anti-REST (Bioss Antibodies, Woburn, MA, Cat. #bs-2590R).