Spe confocal microscope

Cultured cells on coverslips were fixed with 1% or 4% PFA/PBS for 10 min at 22°C and used immediately or stored in PBS at 4°C. Tissue was fixed in 4% PFA/PBS for 10 min at 22°C and equilibrated in 30% sucrose overnight at 4°C. Tissue was embedded in O.C.T. (TissueTek) and stored at –80°C. Sections were cut at 10 μm using a Leica CM3050S cryostat and stored at –80°C. Fixed cells or tissue sections were incubated with primary antibodies overnight at 4°C. Primary antibodies were mouse anti-DDK/FLAG, mouse anti-HSP60, rabbit anti-TH, and rat anti-CD31, all used at 1:500. For kidney sections, fluorescein-labeled Lotus tetragonolobus lectin (LTL) was added during the primary antibody treatment. Incubation with secondary antibodies (1:250) conjugated to either Alexa Fluor 488, Alexa Fluor 555 (Life Technologies), or Cy3 (Jackson ImmunoResearch) was 45 min at 22°C. Samples were then incubated with DAPI (1 ng/ml, Life Technologies) for 5 min at 22°C and mounted in Mowiol 4-88 (Polysciences) with DABCO (25 mg/ml, Sigma-Aldrich). Samples were imaged using a Leica SPE confocal microscope for cell culture and a Zeiss Axio Observer D1 widefield inverted microscope for tissue sections. Quantification of TH-positive cells was performed on 1/3 of the total CB using 1 of 3 sets of adjacent sections (Figure 1G–I).