A00168

Sections were permeabilized for 1 hour in a 4% donkey serum/PBS blocking buffer containing 0,3% Triton X-100 (MilliporeSigma) and incubated overnight at 4°C with the following primary antibodies diluted in a 1% Donkey serum/PBS buffer: rabbit anti-TH (Abcam, EP1532Y, ab137869, 1:2,000) and goat anti-HA (Genscript, A00168, 1:500). Following incubation with primary antibodies, tissues were washed with PBS 3 times for 10 minutes and incubated for 1.5 hours at room temperature with a combination of corresponding donkey anti-species IgG conjugated to AlexaFluor probe (Invitrogen, 1:400). Tissues were then washed with PBS and mounted in DAPI-containing mounting media (Vectashield). Illustrative images were acquired using a confocal microscope Leica SP8 at the BioImaging Center in Bordeaux.

For IF and IEM labelling: mouse anti-HA (1:400, 901502, Covance); mouse anti-β1 integrin (1:200, JB1A, Millipore); mouse anti-α5 integrin (1:200, VC5, BD Biosciences); goat anti-HA (1:400, A00168, Genscript); rabbit anti-GFP (TP401, Acris); rabbit anti-mCherry (1:400) was made on site as described^{60 (link)}, and rabbit anti-vinculin was from Abcam (1:500, ab73412). For detection of GST fusion proteins, we used mouse anti-GST (1:5000, sc138, Santa Cruz). Rabbit anti-goat IgG (1:500, 7S, NORDIC) or rabbit anti-mouse IgG (1:250, Z0412, Dako) was used to bridge between goat or mouse antibodies and protein-A gold^{61 (link)}. Fluorescent secondary antibodies were obtained from Invitrogen (1:250). For immunoprecipitation and western blotting we used mouse anti-GFP (1:2000, 11814460001, Roche), mouse anti-HA (1:2000, 901502, Covance), rabbit anti-mCherry (1:2000) and rabbit anti-GFP (1:2000, ab290, Abcam). Fluorescent secondary antibodies were obtained from Li-Cor (1:20,000).