Rabbit anti glun2b

The protein concentration in the tissues was determined with the Bradford assay kit (Bio-Rad, Hercules, CA, USA). The total and synaptic (Syn) protein extraction (Syn-PER, Thermo Scientific, Waltham, MA, USA) was performed as previously described [19 (link)]. The protein lysates were separated by SDS-PAGE (about 10–12%, sodium dodecyl sulfate, polyacrylamide gel electrophoresis) and transferred to PVDF (polyvinylidene fluoride) membranes. The membranes were blocked in 5% BSA (bovine serum albumin), then incubated with primary antibodies at 4 °C overnight, next incubated off light at RT (room temperature) in secondary antibodies for 1 h (IRD 800 cw, goat-rabbit or goat-mouse 1:10,000; LI-COR). Then, the fluorescence was detected by Odyssey Sa image system (LI-COR) and the densitometric readings were analyzed by Image J software. All antibodies used are as follows: mouse anti-β-actin (1:50,000, Sigma), mouse anti-tubulin (1:50,000, Sigma, Darmstadt, Germany), mouse anti-Reelin (1:500, Milipore, Burlington, MA, USA), rabbit anti-PSD-95 (postsynaptic density protein) (1:2000, Millipore), and rabbit anti-GluN2B, anti-GluN2A, and anti-phosphorylation-GluN2B,(1:1000, Abcam, Cambridge, UK), respectively.

Paraffin sections were dewaxed, rehydrated, antigen repaired (EDTA, Solarbio, C1034, China), and sealed with 5% goat serum (Solarbio, SL038, China) at room temperature for 30 min. The following primary antibodies were used: Rabbit anti-GluN2B (65783, 1 : 100, Abcam, USA); Rabbit anti-m-calpain (39165, 1 : 100, Abcam, USA); and Mouse anti-NeuN (104224, 1 : 100, Abcam, USA). The secondary antibody used was PE-labeled Goat Anti-Rabbit IgG (1 : 200, TransGen, China) and AF488-labeled Goat Anti-Mouse IgG (1 : 200, TransGen, China). The nuclei were labeled after incubated away from the light with DAPI for 2 min. Finally, images of sections were captured using a fluorescence microscope (Leica, Germany).