Immunohistochemistry (IHC) and double IHC

Sections were first rehydrated in PBST (PBS with 0.1% Tween-20), and blocked in 10% fetal bovine serum in PBST for at least 2 hours at room temperature. The primary antibodies were used at different dilutions according to the manufacturer’s instructions and optimization of the protocol (Table 1). The primary antibody incubation was carried out overnight at 4°C in 1% fetal bovine serum in PBST. Sections were then washed 3 times /15 min with PBST and incubated with the secondary antibodies for 2 h at room temperature (Table 1). Sections were again washed with PBST and nuclei visualized with DAPI (Life Technologies, Burlington, ON). The Calbindin, Calretinin, PCNA and TH antibodies required an extra step of antigen retrieval. Sections were treated for 20 min at 85 °C in 0.01 M sodium citrate/0.05% Tween-20 solution and cooled down to RT for 15 minutes prior to blocking. Images were acquired with either a Nikon A1 Confocal microscope or a Zeiss AxioPhot Fluorescence Microscope and treated with NIS-Elements Advanced Research Software or ImageJ.

n.c.: Not conclusive

- no co-localization observed

+ very scarce co-localization (1–15% of cells present co-localization)

++ 15% to 50% of cells present co-localization

+++ Over 50% of cells present co-localization