Anti bdnf

Brains were homogenized and processed for western blotting as previously described [27 (link)]. Thirty μg of proteins were loaded in each lane and separated followed by transfer to nitrocellulose membranes. The membranes were blocked using 5% non-fat milk in TBST (1% tween 20 in tris buffered saline) and probed with the following antibodies antiBDNF (1:250; abcam; Cambridge, MA), TrkB (1:500, abcam; Cambridge, MA), p75NTR (1:1000, Millipore; Billerica, MA), Nogo-A (1:1000, Millipore; Billerica, MA), CHOP (1:1000) and nNOS (1:1000, Cell Signaling; Danvers, MA), ATF6 (1:500), pJNK (1:1000) and JNK (1:1000, Santa Cruz biotechnologies; Santa Cruz, CA). Expression was assessed by quantification of optical density of respective bands normalized to actin using NIH-image J software.

Five sections of the brain cortex and anterior horns of the spinal cord from each rat were randomly selected and subjected to immunofluorescence staining. The tissue sections were incubated overnight at 4 °C with anti-NF-200 (1:500; Abcam, Cambridge, MA, USA), anti-GAP-43 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-BDNF (1:500; Abcam), anti-NF-κB p65 (1:500; Abcam), Anti-p75NTR (1:500; Abcam), anti-MBP (1:500; Abcam), anti-glial fibrillary acidic protein (GFAP, 1:200, Thermo Fisher Scientific, Waltham, MA, USA), anti-COX-2 (1:1000; BioVision, Milpitas, CA, USA), and anti-CD68 (1:100; Santa Cruz Biotechnology) antibodies, individually. After washing in PBS, the sections were incubated with TRITC/FITC-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. The sections were subsequently mounted on glass slides and coverslipped with Antifade Gel Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL, USA). Stained sections were viewed under a microscope at 200x magnification, and images were taken of three fields per tissue section. Areas stained positively for GFAP, MBP, and CD68 were analyzed using NIH Image software. The numbers of cells stained positively for GAP43, caspase-3, NF-200, BDNF, NF-kB p65, COX-2, Caspase-3, and p75NTR were counted.

The protein samples (50 µg of protein) were prepared from micro-dissected hippocampus and separated by 12% SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes using a Bio-Rad Miniprotein III System wet transfer unit for 1 h at 4 °C. Transfer membranes were then incubated with blocking solution (5% nonfat dried milk dissolved in TBST buffer, pH 7.5) for 1 h at room temperature, then incubated with anti-BDNF (1:1000, Abcam), anti-PKA(1:1000, Imagenex), anti-CREB (1:1000, Cell Signaling), anti-pCREB (Ser133, 1:1000, Cell Signaling), anti-MeCP2 (1:2000, Cell Signaling), anti-pMeCP2 (Ser421, 1:1000, Abgent), anti-TrkB (1:1000, Millipore) and anti-pTrkB (Tyr706, 1:1000, Cell Signaling) overnight at 4 °C. Membranes were incubated with secondary antibodies for 1 hat 4 °C. Signal detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences) and quantitated by using the GS-710 Calibrated Image Densitometer (Bio-Rad).

Immunofluorescence was performed following previous studies in which rats anesthetized to death were immobilized by heart perfusion with 0.9% saline and 4% paraformaldehyde [33 (link)]. After repeated perfusion to the anatomy of the brain tissue after the rat body stiffened, the prefrontal regions, such as the coronal section, in turn in 20%, 25%, and 30% sucrose-fixed dehydration, gel embedding, freezing, the parts with cryostat coronary frozen section (10 microns), membrane breaking, repair, washing with phosphate-buffered saline (PBS) 3 times and sealed with 5% sheep serum. The slices were incubated for 12 h in anti-GluA1 (1:200, Cell Signaling, #13185, Shanghai, China), anti-BDNF (1:200, Abcam, ab108319, Shanghai, China), and anti-PSD-95 (1:200, Cell Signaling, #3450, Shanghai, China) at 4°, then incubated at constant temperature in the second antibody for an hour. Finally, the nuclei were stained with DAPI (Solarbio, C0065, Beijing, China), and the immunofluorescence of the treated specimens was observed by fluorescence microscope (Olympus Corporation, Japan). The secondary antibody used was Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (1:200, Proteintech, SA00006-2, Wuhan, China) and Alexa Fluor 594-conjugated Affinipure Goat Anti-Mouse IgG (1:200, Proteintech, SA00006-3, Wuhan, China).