Psd 95

Protein extracts were adjusted to a final concentration of 2.5 μg/μL in sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 200 mM DTT, 0.02% bromophenol blue, pH 6.8) and subjected to SDS-PAGE electrophoresis as described by Laemmli^{53 (link)}. Briefly, samples (25 μg) were separated using 10% polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% BSA, and incubated with primary antibodies against the following targets: PTEN (54 kDa, 1:1000, #9559 Cell Signaling Technology, Danvers, MA, USA), AKT (55 kDa, 1:2000, #sc-1619 Santa Cruz Biotechnology, Dallas, TX, USA), p-AKT^T308 (60 kDa, 1:750, #4056 Cell Signaling), p-AKT^S473 (60 kDa, 1:750, #550747 BD Biosciences, San Jose, CA, USA), S6 (32 kDa, 1:1000, #2217 Cell Signaling), p-S6 (32 kDa, 1:1000, #5364 Cell Signaling), AMPA (100 kDa, 1:500, #13185 Cell Signaling), NR1 (116 kDa, 1:500, #G8913 Sigma-Aldrich, Saint Louis, MO, USA), NR2a (180 kDa, 1:1000, #4205 Cell Signaling), NR2b (190 kDa, 1:1000, #4207 Cell Signaling), PSD-95 (95 kDa, 1:750, #sc-71933 Santa Cruz), synaptophysin (38 kDa, 1:2000, #4329 Cell Signaling), and β-Actin (42 kDa, 1:10000, #A5441 Sigma-Aldrich). Primary antibodies were diluted in 1% BSA, while secondary antibodies were diluted in 5% BSA (1:2000).

The primary antibodies used in the current study were as follows: p-AKT (sc-514,032), NRF-2 (sc-722), AKT (sc-5298), HO-1 (sc-136,961), BDNF (sc-546), PARP-1 (sc-8007), PSD-95 (sc-71,933), synaptosomal-associated protein23 (SNAP-23) (sc-374,215), TNF-α (sc-52,746), interleukin- (IL-) 1β (sc-32,294), p-NF-κB (sc-136,548), syntaxin (sc-2,736), NF-κB (sc-8008), Iba-1 (sc-32,725), GFAP (sc-33,673), and β-actin (sc-47,778) (Santa Cruz, USA). The secondary antibodies were horseradish peroxidase- (HRP-) conjugated anti-mouse (Ref# W402) and HRP-conjugated anti-rabbit (Ref# W401). For immunofluorescence analysis, secondary goat anti-mouse and goat anti-rabbit (catalogue numbers: Ref# A11029 & Ref# 32732, respectively) were used in the optimized dilution.

The process was similar to IHC. Brain samples were quickly obtained and fixed in a 4% PFA solution. Then, 10-μm-thick hippocampal coronal sections were attached to glass slides, dried for 1 h at RT and stored at − 20 °C until use. Sections were permeabilized with prechilled methanol at -20 °C for 10 min and then blocked with 5% normal goat serum solution (Sigma, USA) at room temperature for 1 h. Subsequently, the sections were incubated overnight with anti-BDNF (1:200, Abcam, UK), Nrf2 (1:100, Proteintech, USA), PSD95 (1:100, Santa Cruz, USA), FtL (1:100, Proteintech, USA), TfR (rabbit, 1:100, Abcam, UK), Tf (rabbit, 1:100, Abcam, UK), NeuN (1:100, Abcam, UK) and Iba1 (1:100, Abcam, UK) primary antibodies at 4 °C. The next day, the sections were incubated with Alexa Fluor 488- or 594-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (1:400, Invitrogen). The cell nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI, Solarbio Life Science, China) for 10 min and then cover-slipped with anti-fluorescence quencher (Dako Denmark). A fluorescence microscope (C2 +, Nikon, Japan) or laser scanning confocal microscope was used to visualize immunofluorescence (IF) images of the slices. ImageJ software (National Institutes of Health, USA) was used to analyse the images.

All chemicals, reagents, and materials were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used in this study were GluR-1 (sc-13152 Santa Cruz, Texas, TX, USA), c-Fos (sc-52 Santa Cruz), Bcl-2 (610539), CREB-1 (sc-186 Santa Cruz), TrkB (ab134155, Cambridge, UK), p-mTOR-Ser2448 (#2971, Cell Signaling Beverly, Category, MA, USA), B-actin (sc-47778 Santa Cruz), BDNF (ab108319, Cambridge, UK), PI3K p85α (sc-423 Santa Cruz), P-AKT (#9275 Cell Signaling Beverly), PSD-95 (sc-32290 Santa Cruz), and CaM K II (05-532, Merck, Darmstadt, Germany). Secondary antibodies (Mouse, Rabbit, and Goat) were obtained from Invitrogen (Carlsbad, CA, USA).

After de-paraffinization of sections, the slides were autoclaved in 0.1M sodium citrate pH 6 for antigen retrieval step. The slides were allowed to cool and washed with PBS twice times. Slides were incubated with 5% normal serum depending upon the source of secondary antibody used. The slides were incubated with primary antibodies at 4°C overnight (PSD95, NR2a, synaptophysin, γ-enolase, p-PI3K) from Santa Cruz Biotechnology, and rabbit monoclonal CRMP2, from cell signaling overnight at 1:100 dilution. Next morning, after washing with PBS, fluorescent labeled secondary antibodies (Santa Cruz Biotechnology) were used for signal amplification in dark chamber, followed by mounted with UltraCruz mounting medium (Santa Cruz Biotechnology). The slides were pictured with Confocal scanning microscopes (Flouview FV 1000, Olympus, Japan) using 40× magnification scale, and analyzed by ImageJ, computer based program. ImageJ software was used to quantitatively determine fluorescence intensity of the same region (frontal cortex and striatum) for all groups by optimizing background of image according to the threshold intensity and analyzes the immunofluorescence intensity at the same threshold intensity for all groups and was expressed as the relative integrated density of the samples relative to the control.

IB was performed as described previously^{35 (link)}. Proteins were separated on a 10% gel and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific Inc., MA) using a wet transfer apparatus (Bio-Rad Laboratories Inc., CA). Each membrane was blocked for 1 h at room temperature and then incubated with primary antibodies against PSD-95 (1:200; Santa Cruz Biotechnology Inc., CA), NR2B (1:2000; Proteintech, Chicago, IL), p-NR2B (Ser1303, 1:1000; Abcam, UK), PSD-95 (1:2000; Abcam), p-PSD-95 (Ser295, 1:5000; Abcam), CREB (1:500; Cell Signaling Technology, MA), p-CREB (Ser133, 1:2000; Millipore, CA), tubulin (1:2000; Beyotime Institute of Biotechnology), or GAPDH (1:10000; Aksomics Inc., China) overnight at 4 °C. The blots were then washed and incubated with a peroxide-conjugated secondary antibody (1:2000; Beyotime Institute of Biotechnology) for 2 h at room temperature. Protein bands were visualized by chemiluminescence using an enhanced chemiluminescence reagent (Thermo Fisher Scientific Inc.) and captured using a ChemiDoc MP System (Bio-Rad Laboratories Inc., CA). tubulin was used as a loading control. Commercial markers (Thermo Fisher Scientific Inc.) were used as molecular weight standards.