Ab17345

Western blot analysis was performed as previously described [28 (link)]. Briefly, hippocampal tissue was homogenized. The amount of total protein in the hippocampus tissue lysate was measured according to the Bradford protocol [29 (link)]. After electrophoresis, proteins were transferred from the gel to a polyvinylidene difluoride membrane. Primary antibodies anti-GluN1 (1:1000; rabbit polyclonal ab17345, Abcam, Cambridge, UK) and anti-actin (1:5000; rabbit polyclonal A5060, Sigma-Aldrich, St. Louis, MO, USA) were used. Immunoreactivity was detected with secondary goat anti-rabbit IgG (1:5000; ab6721, Abcam) bound to horseradish peroxidase. Bands were visualized by chemiluminescence method using the Optiblot ECL Ultra Detect Kit (Abcam, ab133409). The relative band intensities were measured by densitometry using Image Lab software (Bio-Rad Laboratories Inc., Hercules, CA, USA). For each sample, the ratio of GluN1 to actin band intensity was calculated.

The proximity ligation assay was performed using the Duolink In Situ Kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's instructions with the following modifications: PLA probe incubation was of 2 h; amplification step was extended to 2 h. Blocking (1 h at room temperature) and primary antibody (overnight at 4 °C) incubations were performed in a 4% bovine serum albumin (Sigma Aldrich) and 0.2% Triton X-100 solution. Rabbit anti-GluN1 (ab17345, Abcam, Cambridge, MA, USA) and rat anti-EGFR (ab231, Abcam) were diluted (1 : 2000 and 1 : 100, respectively) in the blocking solution. The anti-rabbit (+) PLA probe (1 : 5) along with an anti-rat (−) probe (1 : 100) were diluted in the blocking solution. A Goat anti-rat (Jackson ImmunoResearch Inc., Suffok, UK) were used to make a probe anti-rat according to the manufacturer's instructions using the Duolink Probemaker (Olink Bioscience). Slides were mounted in a mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) and 0.1% deparaphenylene-diamine diluted in phosphate-buffered saline and glycerin. The negative control represents the PLA without the primary antibodies.
Ten stack of picture (0.40 μm per section) were taken from 10 different areas of every well with a confocal microscope (Leica SP5, Leica, Nanterre, France). Punctuas were counted manually using a z projection of the stack.