Vectashield antifading mounting medium with dapi

SH-SY5Y cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature followed by sequential incubation with permeabilizing solution (0.2% Triton X-100) in PBS for 30 minutes at room temperature. Then, cultures were washed again with PBS and incubated in blocking solution (3% BSA in 0.5% Tween 20 in PBS) for 30 minutes. Cells were incubated with rabbit polyclonal antibody against TH (1 : 200 dilution in blocking solution; Merck Millipore AB152) overnight at 4°C. After washing, cells were incubated with 1 : 500 dilution of Alexa 488-conjugate secondary antibody for 1 hour at room temperature. Coverslips were then mounted with Vectashield antifading mounting medium with DAPI (Vector laboratories, CA). Cells were visualized under a confocal laser-scanning microscope (Olympus model FV 1000; Tokyo, Japan).

SH-SY5Y cells were plated on 12 mm poly-L-lysine/laminin-coated coverslips at a density of 1 × 10⁴ cells/coverslip. Cells were treated with 10 μM of RA for 4 and 7 days. Cells were then fixed in 4% paraformaldehyde for 15 minutes at room temperature followed by sequential incubation with permeabilizing solution (0.2% Triton X-100) in PBS for 30 minutes at room temperature. Then, cultures were washed again with PBS and incubated in blocking solution (3% BSA in 0.5% Tween 20 in PBS) for 30 minutes. Cells were incubated with rabbit polyclonal antibody against TH (1 : 200 dilution in blocking solution; Merck Millipore AB152) overnight at 4°C. After washing, cells were incubated with 1 : 500 dilution of Alexa 488-conjugate secondary antibody for 1 hour at room temperature. Coverslips were then mounted with Vectashield antifading mounting medium with DAPI (Vector Laboratories, CA). Cells were visualized under a confocal laser-scanning microscope (Olympus Model FV 1000, Tokyo, Japan).

Reactive microglia and proliferating NG2 cells were investigated by double immunofluorescence stainings. The following primary antibodies were used: rabbit anti-NG2 (1:500, Millipore, Temecula, USA), rabbit anti-Iba-1 (1:200; Wako, Neuss, Germany) mouse-anti- RT1B against MHC II (1:500, AbDSerotec, Oxford, UK) and rabbit anti Ki67 (1:200, Abcam, Cambridge, UK). The latter antibody is directed against the endogenous proliferation marker Ki67 [25] (link) and stain cells dividing at the time point of perfusion, which is of importance when assessing alteration in ongoing cell proliferation at different time points [26] (link). Sections were incubated with the primary antibodies (Iba-1/MHCII and NG2/ki67) overnight at 4°C and were the following day incubated with Alexa 488 conjugated goat anti-rabbit (1:200; Vector BA-1000, Vector Laboratories Inc., Burlingame, CA, USA) and Cy3 conjugated horse anti-mouse (1:200; Jackson Immunoresearch, West Grove, PA, USA) for 2 hours in room temperature (RT). Thereafter the sections were mounted in Vectashield anti-fading mounting medium with DAPI (Vector Laboratories, Burlingame, CA).
Alterations in NG2 immunoreactivity were investigated using immunohistochemistry (IHC).