Fv1200 ix83 confocal system

Transduced splenocytes (2 × 10⁶ to 5 × 10⁶) were sorted (live B220⁺ CD138⁻ GFP⁺) and fixed in 2% paraformaldehyde for 40 min on ice. After washing, cells were incubated in 0.5% Triton X-100 for 30 min at room temperature for permeabilization. Primary stain with goat anti-Myc Ab (ab9132; Abcam) and secondary stain with donkey anti-goat Alex-594 Ab (ab150132; Abcam) were performed at room temperature for 45 min to 1 h. After staining, cells were imaged using an Olympus FV1200 IX83 Confocal System, with the use of a ×40 objective.

Organoid samples were fixed with 4% paraformaldehyde, permeabilized using ice-cold methanol, blocked with 10% goat serum for 1 hour at room temperatire, and stained with the following primary antibodies overnight at 4°C: anti-E-cadherin clone 36/E-cadherin, (610181, BD Biosciences), anti-Phospho-Stat3 (Tyr705) clone D3A7, (9145, Cell Signalling), and anti-LGR5 (21833-1-AP, Proteintech). Goat anti-mouse IgG conjugated with Alexa Fluor 488 (A11001, Life Technologies) and goat anti-rabbit IgG conjugated with Alexa Fluor 555 (A32732, Life Technologies) were used as secondary antibodies. Nuclei were stained with Hoechst 33258 (Life Technologies). Images were acquired on an Olympus FV1200 IX83 Confocal System.