Anti fade mounting medium with dapi

Cells were cultured in chamber slides overnight and fixed in 4% paraformaldehyde for 15 min at 4 °C, permeabilized with 0.5% Triton X-100 for 15 min. Cells were then blocked with 5% BSA in PBS and 0.1% Tween-20 (TBST) for 1 h. After the incubation with primary antibodies overnight at 4 °C, cells were then further incubated with anti-mouse IgG (H⁺L), F(ab′)₂ fragment (Alexa Fluor 488 Conjugate, A21206, Life Technologies) or anti-rabbit IgG (H⁺L), F(ab′)₂ fragment (Alexa Fluor 594 Conjugate, A21207, Life Technologies) for 1 h at room temperature. Nuclei were stained with anti-fade mounting medium with DAPI (Invitrogen). Confocal fluorescence images were captured using a Zeiss LSM 710 laser microscope. In all cases, optical sections were obtained through the middle planes of the nuclei, as determined with use of nuclear counterstaining.

Cell slides were prepared before the experiment. After 8 h, the slides containing cells that had migrated were immersed in PBS three times for 3 min each time. The scaffolds were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 at room temperature for 15 min, and sealed with 5% BSA for 1 h at room temperature. The blocking solution was absorbed by the absorbent paper, and a sufficient amount of diluted primary antibody was added to each slide and incubated in a humidified box overnight at 4 °C. The next day, the slides were washed 3 times with TBST, and a diluted fluorescent secondary antibody was added, followed by incubation at room temperature for 1 h in the dark. The slides were washed 3 times using TBST and sealed using 5 μL of antifade mounting medium with DAPI (Invitrogen). Images were acquired under a fluorescence microscope.

A total of 8 × 10⁴ INS-1 832/13 β-cells were seeded in 24-well plates on glass slides and cultured in RPMI 1640, 11 mM glucose, and 10% FBS. The media were switched to 5 mM glucose and after 16 h incubation, INS-1 832/13 β-cells were washed and a secretion medium (HBSS) containing 3 mM glucose was added to the cultures. After 2 h, cells underwent GSIS and were exposed or not exposed to 17 μM HtyOle or 21 μM TyOle, as described. Samples were then washed 2 times with PBS and incubated with a primary anti-insulin antibody (1:200, Dako, Agilent Technologies, Santa Clara, CA, USA) overnight at 4 °C, followed by three 5 min washes in PBS. Dylight 488 secondary Ab (Jackson Immunoresearch, West Grove, PA, USA) incubation was conducted for 1 h at room temperature. Samples were then washed three times and coverslips were mounted using an antifade mounting medium with DAPI (Life Technologies, CA, USA). Images were viewed at 40× magnification and captured through Leica AF6000 microscope (Leica, Wetzlar, German).

The frozen tumor sections were adjusted to RT and then flooded with ice-cold acetone for 10 minutes to fix the tissue. The slides were then air-dried and rehydrated in 0.5% Tween-PBS (PBST) for 3 minutes. The non-specific binding of the secondary antibody was blocked with serum-free protein block (Dako X0909) at RT for 30 minutes and incubated with the following antibodies at 1:25 dilution: F4/80 Alexa Fluor 488 (Bio-rad); 1:50 dilutions: NOS2 (Abcam), CD3 APC (Tonbo Bioscience), CA9 (Abcam), CD68 (Abcam) and TRIB1 (Millipore); 1:100 dilutions: CD31 Alexa Fluor 674 (Biolegend), MR (Abcam), CD4 Alexa Fluor 488 (Biolegend), CD8 PE (Biolegend), IL-15 (Abcam) for 1 hour at RT. The samples were washed twice with PBST for 5 minutes and then incubated with secondary antibody Goat anti-Rabbit IgG (H&L) Dylight 550 (ImmunoReagents), both at 1:50 dilution, Alexa Fluor 488 or 594 goat anti-mouse-IgG or anti-rabbit-IgG secondary antibodies (Invitrogen) at 1:1000 for human TNBC sample, for 1 hour at RT. Slides were washed three times with PBST for 5 minutes and mounted with Antifade mounting medium with DAPI (Life Technology). Slides were kept in the dark overnight at RT and imaged immediately or stored at 4 °C. Random areas of 4-5 images were captured using a Leica AF6000 microscope, or Nikon A1 confocal microscope and cells were manually quantified with ImageJ (NIH).