Mab378

We used a rabbit polyclonal antibody directed against a region near the C-terminal of ApoER2 (A3481, Sigma). The rabbit polyclonal antiserum against the recombinant human ApoER2 cytoplasmic domain and the mouse monoclonal anti-HA have been described before [101 (link)]. We also used a p75^NTR rabbit polyclonal antibody (07–476, Millipore), a mouse monoclonal anti-β-tubulin antibody (05–661, Millipore), a mouse monoclonal anti-actin antibody (MAB1501R, Chemicon), a rabbit polyclonal anti-AKT antibody (#9272, Cell Signaling), a rabbit monoclonal anti-phosphorylated AKT antibody (#4060, Cell Signaling), a mouse monoclonal anti-phosphorylated ERK antibody (sc-7383, Santa Cruz Biotechnology), a rabbit polyclonal anti-Dab1 antibody (AB5840, Chemicon International), and a mouse monoclonal anti-MAP2 antibody (MAB378, Chemicon). We used horseradish peroxidase (HRP)-conjugated secondary antibodies (Chemicon) and Alexa 555- and 488-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Molecular Probes).

Hippocampal primary neurons from albino outbred mice were fixed with fixation solution (4 % formaldehyde in PBS, pH 7.4) for 30 min at room temperature, washed three times with PBS with 0.05 % Tween-20 then incubated at room temperature for 1 h in 5 % BSA in PBS buffer. Primary antibodies anti-map2 mAb (1:1000, Chemicon, mAB378) and anti-synapsin I (1:1000, Chemicon, Temecula, CA) were diluted in 2.5 % BSA in PBS with 0.025 % Tween-20 and incubated at room temperature for 3 h. After three times wash, the hippocampal cultures were incubated in 2.5 % BSA in PBS solution with the secondary antibody (1:1000, Alexa Fluor 488 or 594, Invitrogen) for 1.5 h at room temperature and visualized by a confocal microscope (Thorlabs, Russia). In order to compare data from different culture experiments mean fluorescent intensity of Channel A (synapsin) was divided by the mean fluorescent intensity of Channel B (MAP2).

Neurons were fixed for 10 min with 4% paraformaldehyde in phosphate-buffered saline (ThermoFisher Scientific, #J61899-AP, USA) and stained as previously described (32 (link)). In brief, neurons were immunolabeled using primary antibodies against MAP2ab (Millipore, MAB378, USA; 1:500) with secondary antibodies conjugated to goat-anti-mouse Alexa 488 (ThermoFisher Scientific, #O-6380, USA; 1:1,000) diluted in PBS (ThermoFisher Scientific, #20012043). Nuclei of cells were stained using Hoechst 33342 (3 min; ThermoFisher Scientific, #H3570, USA) and coverslips were mounted using Prolong Gold (ThermoFisher Scientific, #P36930, USA). Z-stack images were obtained using ZEN 2010 Blue Edition software (Zeiss, Thornwood, NY, USA) with a Zeiss LSM 700 laser scanning confocal microscope using a 63x immersion objective (Zeiss, Thornwood, NY, USA). Dendritic branching complexity (e.g., maximum process length and distance from soma with maximal branching) and soma area were assessed with orthogonal projections from Z-stack images using the Sholl analysis tool within ImageJ software [Version 2.1.0; (63 (link))].

After the labelling procedure, the hippocampal or cerebellar slices were fixed with 4% paraformaldehyde at r.t. for 3 h. This was followed by permeabilization and blocking with PBS containing 2% bovine serum albumin, 2% normal goat serum 0.2% triton for 30 min. If these steps are insufficient, fluorescence may be remained in cell bodies. Then, primary antibody reaction was conducted with a rabbit anti-GluA2/3 (Millipore, AB1506, × 500) or a mouse anti-MAP2 (Millipore, MAB378, × 1,000) in PBS at r.t. for 12 h. Secondary antibody reaction was conducted with an Alexa546 anti-rabbit (Invitrogen, A11071, × 1,000) or Dylite650 anti-mouse (abcam, ab96784, × 1,000) in PBS at r.t. for 1 h. The z-stack images of the stained slices were taken using a confocal microscopy (LSM710, Axio Observer.Z1, ZEISS) or two-photon microscopy (LSM780 NLO, Axio Observer.Z1, ZEISS) with 10x objective (NA=0.45) or 63 × oil objective (NA=1.40). Fluorescence images were acquired using a 488 nm line of an argon laser for excitation of Fluorescein, DPPS laser for excitation of Alexa546 and HeNe laser for excitation of Dylite650. CAM2 reagents tend to be incorporated into dead cells. Thus, acutely prepared brain slices should be maintained in a healthy state for visualizing surface-exposed AMPARs.

Cells were fixed and permeabilized with 80% acetone at 4°C for 30 min followed by a blocking step with PBS 1X–10% SVF for 20 min at room temperature. Cells were washed 3 times in PBS 1X and primary antibodies were incubated 1 h at room temperature. To stain both neuronal and glial cells present in the culture, we used a mouse anti-MAP2 antibody (at a concentration of 1/1000) (MAB378, Millipore) and a rabbit anti-GFAP antibody (at a concentration of 1/1000) (AB5804, Millipore), respectively. Upon incubation with primary antibodies, cells were washed 3 times with PBS 1X and incubated with secondary goat anti-mouse antibody coupled to DyLight 549 (at a concentration of 1/1000) (072-04-18-03, KPL) or goat anti-rabbit antibody coupled to DyLight405 (072-08-15-06, KPL), for 1 h at room temperature. For controlling infection, we then used the Light Diagnostics Rabies DFA Reagent (at a concentration of 1/60) (Millipore) for 1 h at room temperature. 15 minutes before the end, TO-PRO-3 (ThermoFisher Scientific) was added to stain cell nuclei and 3 final washes were performed. Images were acquired using aa Zeiss Axioplan fluorescence microscope equipped with a Zeiss ApoTome system (obj.10X).