Nis elements c imaging software

Purified B cells were suspended in 100 μl of RPMI (0.2 × 10⁶ cells) equilibrated for 1 h at 37°C, stimulated with anti-CD22 F(ab′)₂ epratuzumab-A488 (10 μg/ml) (UCB) or left unstimulated, fixed, and permeabilized as described above. Cells were stained with mouse-anti-human-SHP-1 (BD Bioscience) and, for unstimulated control, with anti-CD22 F(ab′)₂ epratuzumab-A488. Washed cells were stained with the secondary antibodies donkey-anti-mouse-RRX. Finally, cells were centrifuged onto slides and covered with Vectashield HardSet mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, USA) to stain the nucleus. Co-localization/fluorescence overlap of SHP-1/CD22 was evaluated (0 = no co-localization, 1 = all pixels co-localized) using a Nikon A1-Rsi confocal microscope and NIS Elements C imaging software (Nikon, Tokyo, Japan).

Enteroids cultured in eight-well chamber slides (Nunc Laboratory-Tek II, MilliporeSigma, Burlington, MA, USA) were fixed with 4% paraformaldehyde for 10 min at room temperature. After fixation, enteroids were permeabilized with 0.2% Triton X-100 for 10 min and blocked in PBS containing 5% goat serum for 1 h at room temperature prior to incubation overnight with rabbit anti-mouse pan-Akt antibodies (4691, Cell Signaling) or rabbit anti-mouse phospho-Akt antibodies (4060, Cell Signaling) at 1:400 dilutions at 4 °C. Slides were then washed with PBS and incubated with Alexa fluor 568 conjugated goat anti-rabbit secondary antibodies (A11036, Invitrogen) at a 1:500 dilution for 45 min at room temperature. After washing with PBS, enteroid cells were counterstained with Hoechst (Thermo Fisher Scientific, Waltham, MA, USA) for nuclei. Finally, the slides were mounted with Prolong gold anti-fade reagent (Thermo Fisher Scientific). The slides were viewed under a Nikon A1R HD25 confocal microscope system with 40× oil objective lens, and images were acquired with the NIS-Elements C Imaging software (Nikon Instruments, Melville, NY, USA).