Researchers create specific cancer organoid system to study bacterial effects on immunotherapy SciReports
At predetermined time point, organoids were washed twice with PBS and fixed at room temperature for 5 h with 4% PFA , washed with PBS, and placed in 70% ethanol for no more than four days before paraffin embedding. 5 µm sections were stained with Hematoxylin and Eosin . For immunofluorescence staining, first, the slides were deparaffinized and incubated for 1-h with citrate pH 6.0 for antigen retrieval and permeablized with 0.1% v/v Triton-X in PBS for 5 min.
Cells were run through a 40 µm strainer to remove any larger organoid pieces that did not fully digest. Cells were spun at 500 g for 5 min and suspended in cold PBS with 10% FBS. Cells were then spun again, and all groups being stained were suspended in flow staining buffer containing 0.2–4 µg/mL conjugated primary antibody or isotype control . Cells were incubated at room temperature for 45 min in the dark. Cells were spun again and washed 3 times with cold PBS with 10% FBS.
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