Polyclonal rabbit anti mouse immunoglobulins fitc

Cells were fixed in either PBS containing 4% paraformaldehyde for 10 min at room temperature or 70% ethanol for 30 min at 4 °C. After washing with PBS, cells were incubated overnight at 4 °C with a mouse monoclonal anti-human 3β-HSD antibody (37-2) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-dopamine D₁ receptor antibody (1:500; abcam, Cambridge, UK) and mouse monoclonal anti-D₂ receptor antibody (1:50; Santa Cruz Biotechnology). In the chemical screening, Polyclonal Rabbit Anti-Mouse Immunoglobulins/FITC (1:200; Dako, Santa Clara, CA) were used as the secondary antibody. For immunostaining of dopamine receptors, Goat Polyclonal Anti-Mouse IgG (Alexa Flour 555, 1:500; abcam) and Donkey Anti-Rabbit Polyclonal IgG (Alexa Flour 647, 1:500; abcam) were used as secondary antibodies. They were incubated for 1 h at room temperature, and then nuclei were visualised with DAPI.

Neurite extension was studied as an in vitro proxy to determine whether collagen-GMA hydrogel could support spinal cord tissue regeneration. Cell-laden gels were incubated in the presence of recombinant human β-NGF (BioLegend, San Diego, CA, USA), added to the media at 100 ng mL⁻¹. Media was changed every 2–3 days. After 7 days, cells were fixed by immersing in 10% neutral buffered formalin (Sigma-Aldrich, Gillingham, UK) for 15 min, and permeabilised by immersing in 0.2% Triton X-100 (Sigma-Aldrich, Gillingham, UK) for 20 min. Samples were stained using Mouse Anti-Neuron-specific β-III Tubulin Monoclonal Antibody (clone TuJ-1) (MAB1195, Bio-Techne, Minneapolis, MN, USA) for 1 h at room temperature, followed by Polyclonal Rabbit Anti-Mouse Immunoglobulins/FITC (F0232, Dako, Glostrup, Denmark) for 1 h at room temperature in the dark. Cells were imaged using a Leica SP8 Confocal Laser Scanning Microscope.