L5626

The sections were dewaxed, completely rehydrated and for antigen retrieval boiled in sodium citrate 0.1 M pH 6. The sections were washed in phosphate buffer solution (PBT, Na₂HPO₄·12H₂O 0.8 M, Panreac 141678.1214, NaCl 0.15 M, Panreac 121659.1211 and 0.075% Triton-X100, Sigma X100) and incubated in PBT with 1.5% H₂O₂ for 30 min to inactivate endogenous peroxidase. After inactivation, tissue was washed in PBT and blocked 1 h in PBT with 0.1% albumin bovine serum (BSA, A2153, Sigma) and 10% lysine 1 M (L5626, Sigma). Next, sections were incubated overnight at room temperature in PBT with 0.1% BSA and 0.01% sodium azide (S2002, Sigma) with different antibodies: αCaspase3 (1:300, Cell Signaling #9661), αChAT (1:100, Chemicon AB144P), αβGalactosidase (1:500, Abcam ab9361) and αTH (1:1000, Inst.J.Boy 28020234). The day after, the tissue was rinsed in PBT and incubated 1 h with the appropriate biotinylated secondary antibody at 1:200. Afterwards, the sections were washed in PBT and incubated in PBT with Avidin–Biotin Complex (1:500, Vectastain PK-4000) for 1 h. Finally, tissue was washed in PBT and Tris 0.1 M pH 7 and the immunolabeling was revealed in Tris 0.1 M with 1% 3-3’ diaminobenzidine tetrahydroc (DAB, Acros Organics W0572M) and 0.003% H₂O₂ leading to a brown precipitate.

The sections were dewaxed, completely rehydrated and for antigen retrieval boiled in sodium citrate 0.1 M pH 6. The sections were washed in PBS-T and incubated in PBS-T with 1.5% H₂O₂ for 30 min to inactivate endogenous peroxidase. After inactivation, tissue was washed in PBT and blocked 1 h in PBS-T with 0.1% albumin bovine serum (BSA, A2153, Sigma) and 10% lysine 1 M (L5626, Sigma). Next, sections were incubated overnight at room temperature in PBS-T with 0.1% BSA and 0.01% sodium azide (S2002, Sigma) with αNKX2.2 (1:5, raised in mouse, Hybridoma Bank, 74.5A5) or αPAX6 (1:200, raised in rabbit, Covance, PRB-278P). The day after, the tissue was rinsed in PBS-T and incubated 1 h with αMouse (1:200, raised in goat, Vector Labs, BA-2020) or αRabbit (1:200, raised in goat, Vector Labs, BA-1000) biotinylated secondary antibody. Afterwards, the sections were washed in PBS-T and incubated in PBS-T with Avidin–Biotin Complex (1:500, Vectastain PK-4000) for 1 h. Finally, tissue was washed in PBS-T and Tris 0.1 M pH 7 and the immunolabeling was revealed in Tris 0.1 M with 1% 3-3′ diaminobenzidine tetrahydroc (DAB, Acros Organics W0572M) and 0.003% H₂O₂ leading to a brown precipitate (0.025% Ammonium Nickel Sulphate was added to obtain black precipitate).