Anti flag fitc

After protein treatment of HeLa cells on a four-well LabTek II chamber slide, the samples were washed with PBS and fixed with fixation solution (1% of methyl alcohol in formaldehyde) for 10 min at RT. Following PBS wash, the samples were treated with permeabilization solution (0.2% Triton X-100 in PBS) for 10 min at RT, and the remaining triton X-100 was removed by PBS wash. The samples were incubated with blocking solution (1% BSA in PBS) for 1 h at RT. Also, the delivered proteins were captured with anti-FLAG FITC (Sigma-Aldrich). After PBS wash, DAPI solution (Sigma-Aldrich) was treated for nuclear counterstaining. The samples were imaged with a confocal microscope (LSM 880, Zeiss).

Pancreas tissue was harvested from BL/6 and BL/6-CD3FLAGmIR mice and fixed in 10% w/v formalin (Thermo Fisher Scientific, Pittsburg, PA, USA) at 4 °C overnight. Slides were de-paraffinized and rehydrated before antigen retrieval, by microwaving in 10 mM citrate buffer pH 6.0. Tissue sections were marked with the hydrophobic barrier. Section was covered with 100 µL blocking buffer, 1% Bovine Serum Albumin in PBS (BSA, Sigma, St. Louis, MO, USA). Sections were stained overnight at 4 °C with anti-FLAG FITC (1:100 Sigma, St. Louis, MO, USA), anti-CD3 APC (1:100, eBioscience, San Diego, CA, USA), guinea pig anti-insulin (1:1500, Sigma, St. Louis, MO, USA), washed and stained with secondary anti-guinea pig IgG Texas Red (1:500, Invitrogen/ Thermo Fisher Scientific Pittsburgh, PA, USA) for 1–2 h at RT. Primary and secondary antibody solution was prepared in the blocking buffer. All staining was performed in humidity chamber to avoid tissue drying and solution evaporation. After washing, sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-Fluoromount G (Sigma, St. Louis, MO, USA) following dehydration and imaged on a Nikon TS fluorescent microscope (Nikon Instruments, Melville, NY, USA).