Rabbit anti map2

Cells were cultured on poly-D-lysine-coated glass coverslips, fixed in 3.7% paraformaldehyde for 20 min at 4°C, and incubated in blocking buffer (PBS containing 10% normal goat serum, 0.4% Triton X-100) for 1 h at room temperature (RT). The primary antibodies rabbit anti-MAP2 (1:400; Santa Cruz Biotechnology), mouse anti-NeuN (1:500; EMD Millipore) and mouse anti-TUJ1 (1:2000; Covance) were applied for overnight incubation. The secondary antibodies mouse Cy3 and rabbit Cy2 (Jackson ImmunoResearch, West Grove, PA, United States) were applied at 1:500 dilution, for 1 h at RT. The samples were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, United States) and analyzed with a Leica fluorescence microscope. The slides were photographed with an ORCA-Flash 4.0 digital color camera and the images processed with the ImageJ software (version 1.47v¹).

Cultured hippocampal cells were incubated with hMSC-EVs (6 × 10⁸ particles) for 22 h after addition of vehicle (PBS containing 2% DMSO) or AβOs (500 nM) for 2 h. In uptake experiments, we employed hMSC-EVs derived from hMSCs that were double-labeled with SYTO RNASelect (for RNA labeling) and Vybrant DiI (for membrane labeling) (both from Molecular Probes). Cells were fixed with 4% paraformaldehyde/4% sucrose solution for 15 min at 4 °C. Cell nuclei were labeled with DAPI. Images were acquired on a Zeiss LSM 510 confocal microscope.
For immunocytochemistry, fixed cells were blocked with 4% bovine serum albumin at room temperature. Primary antibodies used were rabbit anti-MAP 2 (1:200; Santa Cruz), mouse anti-GLUA1 (1:200; MAB2263, Santa Cruz), and guinea pig anti-GLT-1 (1:400; AB1783, Millipore) and were incubated overnight at 4 °C. For labeling with mouse anti-synaptophysin (1:1000; Vector Labs) and rabbit anti-PSD-95 (1:200; Santa Cruz) antibodies, cells were permeabilized with 0.1% Triton X-100 (Merck) for 5 min at room temperature before blocking with 10% horse serum for 1 h. After incubation with primary antibodies, cells were washed with PBS and incubated with Alexa Fluor-conjugated secondary antibodies (1:1000; Thermo Fisher) for 2 h at room temperature. Images were acquired on a Zeiss Axio Observer Z1 microscope or a Nikon C2 confocal microscope.

Neurons (10 DIV) were fixed in 4% paraformaldehyde in PBS for 20 min or, in the case of pan-Nav1 stainings, for 2 min in 2% paraformaldehyde in PBS followed by 10 min in 100% methanol at −20°C. Blocking of unspecific binding was performed for 30 min with 0.2% fish skin gelatin (Sigma-Aldrich) in PBS for surface-staining or in 0.1% Triton X-100 (Sigma) in PBS (PBST) for total-staining for 30 min at room temperature (RT). Next, neurons were incubated with primary antibodies diluted in 0.2% fish skin gelatin in PBS or PBST for 1 h at RT. Lastly the neurons were incubated with secondary antibodies diluted in 0.2% fish skin gelatin in PBS or PBST for 45 min at RT. Primary antibodies used: mouse anti-CD4 (1:25–1:50 dilution; clone 18–46; Santa Cruz Biotechnology), mouse anti-CD4 (1:50 dilution, MT310, Santa Cruz Biotechnology), rabbit anti-MAP2 (1:100 dilution, H-300, Santa Cruz Biotechnology), mouse anti-pan-Nav1 (1:100 dilution, clone N419/40, RRID:AB_2491098, Neuromab), mouse anti-ankG (1:5, clone N106/65, RRID: AB_10673449, Neuromab), mouse anti-GFP (1:5, clone 4C9, Developmental studies Hybridoma Bank). Primary antibodies were detected using AlexaFluor^®-conjugated secondary antibodies (Thermo Fischer Scientific). Coverslips were mounted on microscope slides using ProLong Gold or Diamond Antifade Reagent (Thermo Fischer Scientific).

Cryosections of cholesteatoma tissue and external auditory canal skin or cultivated cells were fixed for 20 min using 4% PFA followed by permeabilization in TritonX-100 (tissue: 0.2%, cells: 0.02%, Applichem) containing 5% goat serum for 30 minutes. Primary antibodies used were mouse anti-Nestin 1:200 (Millipore), rabbit anti-S100B 1:100 (Dako) for stem cell detection already utilized in Hauser et al.^{15 (link)}. Additional primary antibodies were mouse anti-β-III-tubulin 1:100 (Promega), rabbit anti-MAP2 1:100 (Santa Cruz Biotechnology), mouse anti-TLR4 1:100 (Acris Antibodies GmbH), rabbit ant-β1-integrin (Sigma Aldrich), mouse anti-CK-14 1:200 (Millipore) and mouse anti-CK-18 1:800 (Cell Signaling Technology). They were applied for 1 h (cells) or 2 h (sections) at RT. Secondary fluorochrome-conjugated antibody 1:300 (Alexa 555 anti-mouse or Alexa 488 anti-rabbit, Invitrogen, Life Technologies GmbH) were subsequently applied for 1 h at RT. Nuclear counterstaining was performed using 4,6-Diamidin-2-phenylindol (DAPI, 1 µg/ml) for 15 minutes at RT followed by mounting with Mowiol. Imaging and analysis was performed using confocal laser scanning microscope (CLSM 780, Carl Zeiss) with ZEN software (Carl Zeiss).