Alexa fluor 488 goat anti mouse igg

Cx43 was detected using rabbit polyclonal antibodies directed against amino acids 363–382 of human/rat Cx43 (C6219, SIGMA-Aldrich, St. Louis, MO, USA) at 1:5000 dilution for immunoblotting and at 1:250 dilution for immunofluorescence. Mouse monoclonal antibody against human recombinant ZO-1 fusion protein encompassing amino acids 334–634 (cat no 33-9100, Thermo Fisher Scientific Inc.) was diluted 1:250 for immunofluorescence. VE-cadherin was detected using mouse monoclonal antibodies (sc-9989, Santa Cruz, Biotechnology, Inc., Santa Cruz, CA, USA) at 1:100 dilution for immunofluorescence.
AlexaFluor 488 goat anti-mouse IgG and horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA) and used according to manufacturer’s instructions.

SH-SY5Y cells were fixed using 4% paraformaldehyde and 4% sucrose in DPBS (pH 7.4) for 20 min at room temperature (RT). Next, the cells were permeabilized and blocked using DPBS containing 1% BSA and 0.1% Triton X-100 at RT for 30 min, and then primary antibodies (mouse anti-EAAC1 (1:100) and rabbit anti-tau (1:100)) were added and incubated overnight at 4 °C. The cells were then washed three times in PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-chicken IgG (Jackson ImmunoResearch Laboratories, Inc., 1:200) for 2 h at RT. After counterstaining with DAPI (10 μM in DPBS), the cells were mounted in Vectorshield (Vector Laboratories). Fluorescent images were acquired with an LSM 5 LIVE confocal system (Carl Zeiss AG, Oberkochen, Germany).

Quail cultures (grown directly on plastic plates) were fixed by 4% paraformaldehyde (PFA) for 15 min, permeabilized with PBS containing 5% FBS (fetal bovine serum) and 0.1% triton X-100. NeuN or MAP2-immunostaining were performed by overnight incubation of fixed cells with anti-NeuN antibody (mouse, clone A60, Millipore CAT# MAB377) (1:1000 in PBS) or with anti-MAP2 (mouse, cat # MA5-12826, Thermo Fisher) (1:500 in PBS) at 4 °C, with 3% FBS and 0.1% triton X-100. The next day, plates were washed three times with PBS and stained by secondary antibody (Rhodamine Red-X goat Anti-mouse IgG, Jackson Laboratories, CAT# 115-295-003) (1:200), in 3% FBS and 0.1% triton X-100 for 1 h at room temperature. Plates were then washed by PBS and stained with DAPI (1:1000). NeuroTrace 640/660 staining (Thermo Fisher) was performed as previously described in ref. ^{122 (link)}. Briefly, cultures were fixed as described above, permeabilized with PBS containing 0.1% triton X-100 for 10 min, washed, and followed by incubation for 30 min with PBS containing NeuroTrace stain (1:100). Then, cells were washed by PBS and stained with DAPI (1:1000). GFAP-staining (1:500) was performed by using anti-GFAP (mouse, clone G-A-5, Calbiochem, CAT# IF03L), followed by staining with a secondary antibody (1:500) (Alexa Fluor-488 goat anti-mouse IgG, Jackson Laboratories, CAT# 115-545-003).