Mice at between P42–P46 for the adult time point or P10 for the developmental time point (n = 3 or 4 for each condition) were perfused with 4% paraformaldehyde (PFA) and brains were fixed overnight in 4% PFA at 4 °C. 50-μm vibratome sections were used for all histological experiments. Every 4th section was collected for the representation of each brain and the sections were processed for immunohistochemistry in order to confirm somatostatin identity, but also to amplify weak signals that could come from low levels of TVA expression.
For the immunofluorescence, brain sections were incubated 1 h at room temperature in a blocking solution containing 3% Normal Donkey serum and 0.3% Triton X-100 in PBS and incubated overnight or 48hrs at 4°C with primary antibodies: rat anti-RFP (1:1,000; Chromotek #5f8), chicken anti-GFP (1:1,000; Aves Labs #1020), rabbit anti-somatostatin (1:3,000; Peninsula Laboratories International T-4103.0050), goat anti-ChAT (1:250; Millipore AB144P), goat anti-CTGF (1:500; Santa Cruz Biotechnology sc-14939), rabbit anti-TPH2 (1:500, Novus Biologicals NB74555). Sections were rinsed three times in PBS and incubated for 60–90 min at room temperature or overnight at 4°C with the Alexa Fluor 488-, 594- or 647-conjugated secondary antibodies (1:500; Thermo Fisher Science or Jackson ImmunoResearch).
For the immunofluorescence, brain sections were incubated 1 h at room temperature in a blocking solution containing 3% Normal Donkey serum and 0.3% Triton X-100 in PBS and incubated overnight or 48hrs at 4°C with primary antibodies: rat anti-RFP (1:1,000; Chromotek #5f8), chicken anti-GFP (1:1,000; Aves Labs #1020), rabbit anti-somatostatin (1:3,000; Peninsula Laboratories International T-4103.0050), goat anti-ChAT (1:250; Millipore AB144P), goat anti-CTGF (1:500; Santa Cruz Biotechnology sc-14939), rabbit anti-TPH2 (1:500, Novus Biologicals NB74555). Sections were rinsed three times in PBS and incubated for 60–90 min at room temperature or overnight at 4°C with the Alexa Fluor 488-, 594- or 647-conjugated secondary antibodies (1:500; Thermo Fisher Science or Jackson ImmunoResearch).