Immunohistochemistry and Image Quantification

Mice were deeply anesthetized with isoflurane, transcardially perfused with ice-cold PBS solution followed by fresh ice-cold 4% paraformaldehyde (PFA) in PBS. Brains were dissected out and postfixed overnight with 4% PFA at 4°C, and stored in PBS at 4°C. Sixty to hundred microgram sections were cut on a vibratome (Leica VT100S). Floating brain slices were permeabilized with 1.2% Triton X-100 in PBS for 15 min, washed 3 × 5 min in PBS and blocked 1 h in 5% normal goat serum, 2% BSA, 0.2% Triton X-100 in PBS (blocking buffer) for 1 h at room temperature. Sections were incubated with primary antibodies in blocking solution 1–3 overnights at 4°C. Slices were then washed three times with PBS for 20 min, incubated with secondary antibodies for 1–2 h at room temperature, washed 1× with DAPI (1:10,000) in PBS for 20 min then 2× with PBS for 20 min. Slice were mounted with 50% glycerol in Tris Buffer. Primary antibodies used were ms anti-TPH2 (1:500, Sigma, St. Louis, MO, USA to678), ms IgG1 anti-TH (1:1,000, immunostar 22941), rb anti p2A (1:1,000 Millipore, Burlington, MA, USA ABS31), and rat anti-DAT (1:1,000 Millipore, Burlington, MA, USA MAB369). Widefield epifluorescent images were capture using a 4× lens on a Olympus BX61 microscope with a PRIOR ProScan III motorized stage (Prior Scientific Instruments, UK) and CellSens Dimension 1.11 stitching software (Olympus). To quantify overlap, images were captured using Olympus Fluoview FV1000 confocal microscope, 20× lens. Three mice were imaged per condition, three sections per mouse. DAT sections, since they were coronal, were imaged bilaterally. Cells were counted manually using the ImageJ Cell Counter plugin (Schindelin et al., 2012). To quantify cell number and TPH2 and DAT expression levels in old animals over 1 year old, serial 60 um sagittal sections were collected from TPH2 and DAT-eArchT3.0 BAC transgenic animals and immunostained with TPH2, DAT or TH. Every third section from three animals was confocal imaged as described above; sections were matched referenced to the midline. Cells were counted automatically with CellProfiler (Lamprecht et al., 2007) using primary object detection and the Otsu global thresholding method. For the DAT-eArchT3.0 transgenic line, DAT immunostaining did not clearly distinguish individual dopaminergic cell bodies, so TH staining was used. To quantify DAT expression levels, DAT staining in the striatum was used to take advantage of relatively uniform coverage by dopaminergic terminals. To correct for absence of terminals around nuclei and fibers of passage, a mask was used to remove those areas from quantification. For the TPH2 transgenic line, TPH2 immunostaining was used both to quantify cell number and to assess expression levels. To correct for variable cell number between sections and animals, intensity levels were only measured from detected cells and normalized to total cell area per image.