Poly d lysine laminin coated glass coverslips

The cells plated onto poly-D-lysine/laminin-coated glass coverslips (BD Biosciences) were incubated with 4% paraformaldehyde for 20 min. After blocking with 1% BSA in PBS with 0.1% Tween 20 (PBS-T), the cells were incubated with primary antibodies overnight at 4 °C and subsequently with the appropriate secondary antibodies. The samples were mounted in the ProLong Gold Antifade Reagent with DAPI (Invitrogen, Waltham, MA, USA). The results were analyzed using a Nikon epifluorescence microscope (Nikon, Tokyo, Japan).

Cells with or without treatment with EGF at 50 ng/ml for 30 min were plated onto poly-D-lysine/laminin-coated glass coverslips (BD Biosciences), fixed, and permeabilized with 4% formaldehyde in 100 mM PIPES (pH6.8), 10 mM EGTA, 1 mM MgCl₂, and 0.2% Triton-X 100 for 15 min at room temperature. Following PBS washing, cells were blocked in 1% BSA in PBS plus 0.1% Tween 20 (PBS-T) for 30 min and further incubated with primary antibodies overnight at 4°C. After PBS-T washing, cells were incubated with the appropriate secondary antibodies. Samples were mounted in the ProLong Gold Antifade Reagent (Invitrogen), and results were analyzed with a Nikon epifluorescence microscope (Nikon).

The cultures were prepared as described previously (Zhong et al., 2015 (link)). Briefly, for ventral hippocampal micro-slice cultures, the ventral CA1 and subiculum area (vHipp) from WT (+/+) or α7 knockout (−/−) mice (postnatal days 0–3, P0–P3) was dissected, sliced into ∼150 μm × 150 μm pieces, and then plated onto poly-D-lysine/laminin-coated glass coverslips (12 mm, BD Sciences) in 24-well plates with a minimal volume (50 μl) of culture media [Neurobasal, 2% B-27 (GIBCO) and 20 ng/ml of brain-derived neurotrophic factor (Invitrogen)]. After the micro-slices attached to the substrate, 100 μl of additional culture media was added. For medial septum/diagonal band (MS/DB) cultures, the MS/DB area (Bakker et al., 2015 (link); Staib et al., 2018 (link)) from ChAT-tau-eGFP or ChAT-IRS-Cre mice (P0–P3) was dissected, sliced and plated on coverslips as described before (Zhong et al., 2015 (link)) and above for vHipp cultures.
For vHipp-nucleus accumbens (nAcc) synaptic co-cultures, nAcc (ED18-P1) from WT mice (C57BL/6J) were dissected out and dispersed with 0.25% trypsin (GIBCO) for 15 min at 37°C, followed by gentle trituration in culture media. Dispersed nAcc neurons were plated onto poly-D-lysine/laminin-coated glass coverslips at 0.25 ml/coverslip. The next day vHipp micro-explants [prepared as described above and in Zhong et al. (2008) (link)] were added to the same coverslips.

Mouse TG neurons were cultured for calcium imaging experiments. Naïve male and female mice were injected with WGA as described above, bilaterally in the tongue and 2 days later, TGs were dissected for neuronal cultures as described previously^{125 (link)}. Tissues were washed in HBSS and then dissociated with 1 mg/ml of collagenase and dispase (Roche, Indianapolis, IN, USA) for 45 min at 37 degrees. Following washing out the enzymes, cultures were plated on poly-D-lysine/laminin coated glass coverslips (BD Biosciences, San Jose, CA, USA) and grown overnight in either DMEM media supplemented with glutamine, penicillin/streptomycin and 2% Fetal bovine serum (for vehicle group) or the same media with 100 ng/ml recombinant colony-stimulating factor 1 (Csf1, Cell Signaling, Boston, MA, USA). Csf1 stock was diluted in saline.

The cells were grown on poly-D-lysine/laminin-coated glass coverslips (BD Biosciences) in 24-well plates. Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.3% Triton X-100 for 5 min. Following blocking in 1% BSA in PBS plus 0.3% Triton-X for 30 min at room temperature, cells were incubated with the following primary antibodies: p65 (8242S, clone D14E12, dilution 1:1,000, Cell Signaling), β-catenin (sc1496-R, clone C-18, dilution 1:1,000, Santa Cruz Biotechnology), p-Erk1/2 (4370S, clone D13.14.4E, dilution 1:250, Cell Signaling), IκB (4814S, clone L35A5, dilution 1:1,000, Cell Signaling) and p- p65 (S536; 3033S, clone 93H1, dilution 1:1,000, Cell Signaling) at 4 °C overnight. After washing with PBS, cells were incubated with fluorescence-conjugated secondary antibodies at room temperature for 1 h. Cells were mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen) and analysed using a Nikon epifluorescence microscope.