4 6 diamidino 2 phenylindole (dapi)

Deparaffinization and antigen retrieval of the brain tissue sections were performed as previously described.^{30 (link)} After washing with phosphate-buffered saline (PBS), sections were blocked in 0.1% Triton X-100 (Sigma-Aldrich), 2% normal goat serum (Thermo Fisher Scientific, MA, USA) and 1% bovine serum albumin (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies diluted in blocking buffer overnight at +4°C. After 3 × 10 min washes in 0.1% Triton X-100 in PBS, they were incubated with secondary antibodies and DAPI (2-(4-Amidinophenyl)-1H-indole-6-carboxamidine) for 1 h at room temperature followed by 3 × 10 min washes in Triton X-100 in PBS. The following dilutions and antibodies were used: 1:100 mouse anti-phospho-tau (Thr212, Ser214) (MN1060; Thermo Fisher Scientific), 1:100 rabbit anti-TRX-1 (MA532569; Thermo Fisher Scientific), 1:100 rabbit anti-AGT (MA529010; Thermo Fisher Scientific), 1:500 Alexa Fluor fluorescently conjugated secondary goat anti-rabbit and anti-mouse antibodies (Thermo Fisher Scientific) and 1:100 DAPI (1351303; Bio-Rad, USA). To quench the autofluorescence of lipofuscin, sections were washed for 5 min in PBS and incubated with 0.1% w/w Sudan Black B (199664; Sigma-Aldrich) in 70% ethanol. Prior to mounting, sections were washed with Triton X-100 in PBS 2 × 10 min and 10 min with PBS.

Cells were fixed, permeabilized and blocked as previously published [12 (link)] and incubated overnight at 4 °C with primary Abs. Then, cells were incubated for 2 h in the dark with the secondary Abs. Nuclei were counterstained with DAPI (Bio-Rad) for 15 min. Slides were visualized at 64X magnification as previously described [12 (link)]. A minimum of 200 cells per condition was analyzed using the ImageJ program.

Deparaffinization and antigen retrieval of the brain tissue sections were performed as previously described.^{30 (link)} After washing with phosphate-buffered saline (PBS), sections were blocked in 0.1% Triton X-100 (Sigma-Aldrich), 2% normal goat serum (Thermo Fisher Scientific, MA, USA) and 1% bovine serum albumin (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies diluted in blocking buffer overnight at +4°C. After 3 × 10 min washes in 0.1% Triton X-100 in PBS, they were incubated with secondary antibodies and DAPI (2-(4-Amidinophenyl)-1H-indole-6-carboxamidine) for 1 h at room temperature followed by 3 × 10 min washes in Triton X-100 in PBS. The following dilutions and antibodies were used: 1:100 mouse anti-phospho-tau (Thr212, Ser214) (MN1060; Thermo Fisher Scientific), 1:100 rabbit anti-TRX-1 (MA532569; Thermo Fisher Scientific), 1:100 rabbit anti-AGT (MA529010; Thermo Fisher Scientific), 1:500 Alexa Fluor fluorescently conjugated secondary goat anti-rabbit and anti-mouse antibodies (Thermo Fisher Scientific) and 1:100 DAPI (1351303; Bio-Rad, USA). To quench the autofluorescence of lipofuscin, sections were washed for 5 min in PBS and incubated with 0.1% w/w Sudan Black B (199664; Sigma-Aldrich) in 70% ethanol. Prior to mounting, sections were washed with Triton X-100 in PBS 2 × 10 min and 10 min with PBS.

Mice were transcardially perfused and the pancreas was processed as described previously^{12 (link)}. Mounted 16 μm-thick sections were incubated with primary antibodies in a humidified chamber at 4 °C overnight (rabbit anti-glucagon antibody, 1:1,000, BioGenex, Fremont, CA, USA) and guinea pig anti-insulin antibody (1:200, Bio-Yeda, Rehovot, Israel). After washing in PBS (0.01 M) they were incubated with secondary antibodies for 90 min at room temperature (donkey Cy3-conjugated anti-rabbit IgG and donkey FITC-conjugated anti-guinea pig IgG, 1:150 and 1:40 respectively, Jackson Immunoresearch Europe, Suffolk, United Kingdom). Sections were finally incubated with DAPI (1:10,000, Bio-Rad, Hercules, CA, USA) diluted in PBS for 15 min at room temperature and mounted using 2.5% DABCO in glycerol (Sigma-Aldrich, ST. Louis, MO, USA) and stored at −20 °C in darkness. Another set of sections was used for H&E staining (Histolab, Gothenburg, Sweden) and mounted with VectaMount permanent mounting medium (Vector Laboratories Inc., Burlingame, CA, USA). For the detection of apoptotic β cells, sections were processed as according to the commercial kit used (Click-iT Plus TUNEL assay, Thermo Scientific, Waltham, MA) and co-labeled with insulin.