Sc 166940

For immunofluorescence, brain slices were first treated with sodium citrate (0.01 M, pH 6.0) at 95°C for 10 min. After being rinsed with PBS for three times (each for 15 min), brain sections underwent blocking with serum containing 0.5% Triton X-100 for 2 h at room temperature, followed by primary antibody incubation at 4°C overnight. The following primary antibodies were used: goat anti-ChAT (1:500; AB144P; Sigma-Aldrich), rabbit anti-NeuN (1:500; ab177487; Abcam), rabbit anti-Glial fibrillary acidic protein (GFAP; 1:1000; Z0334; Dako), rabbit anti-Cleaved caspase-3 (1:500; 9661s; Cell signaling technology), rabbit anti-Iba1 (1:200; A19776; Abclonal), rabbit anti-alpha-synuclein (phosphor S129) (1:500; EP1536Y; Abcam), mouse anti-Tyrosine hydroxylase (TH; 1:1000; T2928; Sigma-Aldrich), and mouse anti-c-Fos (1:100; sc-166940; Santa Cruz). After primary antibodies incubation, all sections were rinsed with PBS three times (each for 15 min) and stained with species-specific Alexa Fluor 488- or biotin- conjugated secondary antibodies at room temperature for 3 h. Thereafter, Cy3-conjugated streptavidin and Hoechst 33,258 were used for biotin-conjugated secondary antibodies for 1 h at room temperature, and all sections were mounted with 75% glycerol.

Immunohistochemistry (IHC) was performed automatically on a Ventana BenchMark Ultra instrument (Ventana Medical System, Tucson, Arizona, USA). The primary antibody used was c-FOS (E8): sc-166940 (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:600. After deparaffination and rehydration of the sections, antigen retrieval was done using EDTA buffer at 100 °C for 30 min. Slides were treated with inhibitors to endogenous peroxidase followed to incubation with primary antibody in 30 min at 37 °C. Amplification was done using Optiview system (Ventana Medical System, Tucson, Arizona, USA). Visualization was done using Diaminobenzidine (DAB) and Hematoxylin was used as counterstain. Human term placenta was used as positive control.

The sections of pancreatic tissues were blocked by 10% goat serum (Servicebio, Wuhan, China) and incubated with primary antibodies at 4°C overnight. The primary antibodies used in this experiment were: rabbit anti-insulin (1:200; bs-0056R; Bioss), mouse anti-insulin (1:200; BM0080; Boster), rabbit anti-podoplanin (1:200; A01124-2; Boster), mouse anti-Fos (1:100; sc-166940; Santa Cruz), mouse anti-Bad (1:100; sc-8044; Santa Cruz). The secondary antibodies (SA00013-1, SA00013-2, SA00013-3, SA00013-4) for immunostaining were all purchased from Proteintech. 4′,6′-diamidino-2-phenylindole (DAPI; Servicebio) was used for nuclei staining. Olympus FV1000 confocal microscopy system was used for images capture.