Alexa fluor igg h l

For histological examination, organoids were fixed in 4 % PFA overnight at 4 °C, washed with PBS, and pre-embedded in 2% agarose (Sigma) in PBS. After serial dehydration, intestinal organoids were embedded in paraffin, sectioned at 4 µm, deparaffinized, rehydrated, and subjected to heat-mediated antigen retrieval in tris Buffer (pH 9) or citrate buffer (pH 6). Sections were permeabilized with 0.5% Triton-X for 30 min at RT and stained overnight with primary antibodies (rabbit anti-IFITM1 Cell Signaling 13126S, 1:500 or rabbit anti-IFITM2 Cell Signaling #13530S, 1:500 or rabbit anti-IFITM3 Cell Signaling #59212S, 1:250 or anti-SARS-CoV-2 N 1:500 or anti-E-Cadherin 1:500) diluted in antibody diluent (Zytomed) in a wet chamber at 4 °C. After washing with PBS-Tween 20, slides were incubated with secondary antibodies (Alexa Fluor IgG H + L, Invitrogen, 1:500) and 500 ng/ml DAPI in antibody diluent for 90 min in a wet chamber at RT. After washing with PBS-T and water, slides were mounted with Fluoromount-G (Southern Biotech). Negative controls were performed using IgG controls or irrelevant polyclonal serum for polyclonal antibodies, respectively. Cell borders were visualized by E-cadherin staining. Images were acquired using an LSM 710 system.