T-4103

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Publication protocol

For immunohistochemical procedures, PV (Swant, PV 27) and SOM (Peninsula Laboratories, T-4103) antibodies were used. For both the PV and SOM staining, a free-floating standard 3, 3’-diaminobenzidine (DAB) peroxidase protocol was carried out at room temperature. Briefly, the sections were rinsed in 0,1 M PBS for 10 min, followed by a 30-min incubation with 0.3% H2O2. Then, the sections were rinsed three times for 15 min and a 30-min pre-incubation in PBS-BT was performed. The PV (1:40,000) and SOM (1:10,000) antibodies were used as primary antibodies and sections were incubated overnight at room temparature. After the incubation, sections were washed in PBS, followed by 90 min of incubation with Donkey-Anti-Rabbit IgG Biotin SP conjugated (Jackson ImmunoResearch, 1:1,500). Afterwards sections were washed again three times for 15 min and then incubated for 90 min with Vector ABC-Elite (Vector Labs, A and B 1:800) followed by the same washing steps. Then, a pre-incubation with DAB-Ni solution (Vector Labs, without perhydrol) was carried out for 10 min followed by the incubation with DAB-Ni solution with perhydrol. Sections were mounted on gelatin-coated object glasses and dried overnight at 37°C. The sections were then dehydrated in an alcohol series and mounted with Entellan.

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