Cy5 conjugated anti mouse igg

The following primary Abs were employed throughout this study: anti-CD9 clone P1/33/2 (Santa Cruz, Dallas, TX), anti-CD9 clone MEM-61 (Abcam, San Francisco, CA), anti-CD9 clone M-L13 (BD Biosciences, San Jose, CA), anti-CD9 clone HI9a (BioLegend, San Diego, CA), anti-CD81 (cat. #349502, BioLegend), anti-CD63 (cat. #sc-15363, Santa Cruz Biotechnology, Dallas, TX), anti-ADAM-10 (cat. #sc-25578, Santa Cruz Biotechnology), anti-β-actin (cat. #sc-47778, Santa Cruz Biotechnology), anti-IgSF8 (cat. #103561, Santa Cruz Biotechnology), anti-β1 integrin (cat #4706S, Cell Signaling Technology, Danvers, MA), anti-α-tubulin (cat. #sc-8035, Santa Cruz Biotechnology), anti-acetylated-α-tubulin (cat. #T7451, Sigma), anti-CD44 (cat. #MA5-13890, ThermoScientific, Waltham, MA). The following secondary Abs were employed: TRITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch), TRITC-conjugated anti-goat IgG (Sigma), and Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch). For inhibition experiments, sodium azide was removed by desalting through Sephadex G-25 spin columns.

Isoproterenol, SR-58611A, and3-isobutyl-1-methylxanthine(IBMX), were from Sigma–Aldrich (Hackettstown, NJ, USA); mouse anti-HA antibodies were from Biolegend (Berkley, CA, USA); Cy5-conjugated antimouse IgG were from Jackson Immuno-Research Laboratories (West Grove, PA, USA); DNA oligonucleotides were purchased from Macrogen (Seoul, South Korea); pLenti-C-mGFP vector was from Origene (Rockville, MD, USA); restriction enzymes were from Promega (Madison, WA, USA) and Phusion High-Fidelity DNA Polymerase Mix was from Thermofisher (Waltham, MA, USA).

Mice were sacrificed, and pancreas samples were collected and processed as previously described [20 (link)]. 1% Thioflavin S (T1892, Sigma-Aldrich, St. Louis, MO, USA) in ddH₂O was used to stain for amyloid aggregates directly after deparaffinization. For the detection of β-cells, a rabbit mAb [EPR17359] IgG to Insulin (ab181547, Abcam, Cambridge, UK), followed by a goat anti-rabbit IgG conjugated to biotin (Nordic-MUbio, Susteren, The Netherlands) and a streptavidin conjugated Alexa546 (s11225, Molecular Probes, Eugene, OR, USA), were used. mAb IL-1β (F-5, sc-515598, Santa Cruz), followed by a Cy5-conjugated anti-mouse IgG (Jackson ImmunoResearch, Cambridgeshire, UK), was used to identified inflamed cells. For intracellular staining, sections were permeabilized with 0.5%-TritonX-100 in PBS. As a blocking solution bovine serum (3%), casein (0.5%) and NaN₃ (0.1%) were prepared; however, when performing intracellular staining, the blocking buffer was made up of BSA (2%) and 0.5%-Triton X-100. In the presence of biotinylated Abs, an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA; USA) was used to block endogenous biotin according to the manufacturing instructions. All pictures were acquired with an Axio Imager.A2, and a Carl Zeiss AxioCam.
Quantifications were performed using Fiji Image J (NIH, USA), as previously described [20 (link)].