Leica tcs sp8 microscope

A confocal microscope was employed to assess the presence of nisin on the surface of powdery samples loaded with the compound. Nisin fluorescence was generated by utilizing an excitation wavelength of 405 nm, and the emitted light was recorded within the 470–580 nm range. This imaging was performed using a Leica TCS SP8 microscope (Leica Biosystems, Milan, Italy).

To split f-IHOs, Matrigel® was digested with Trypsin-EDTA for 15 min at 37 °C. Cell suspension was centrifuged at 300g for 4 min, washed once with William’s E medium and resuspended in Matrigel® domes as described above. Organoids were typically passaged at a 1:4 ratio. For IF staining of organoids, Matrigel® was dissolved in cell recovery solution (11543560, ThermoFischer scientific) for 30 min at 4 °C and fixed for 30 min in 4% w/v PFA. Blocking was performed for 1 h in 3% donkey serum, 0.3% Triton and 0.1% DMSO. Primary antibodies were used 4 °C overnight at the indicated dilutions: CDH6 (AF2715, sheep, 1:50; RnD systems), EPCAM (ab7504, mouse, 1:100; abcam), KRT7 (ab9021, mouse, 1:100; abcam), HNF4A (ab92378, rabbit, 1:100; abcam), CK19 (ab52625, rabbit, 1:200; abcam), albumin (A80-129A, goat, 1:100; Bethyl), TROP2 (10428-MM02, mouse, 1:100; Stratech). DAPI was used at 1:1000 dilution as a counterstain. Alexa Fluor-conjugated secondary antibodies (all 1:500, Life Technologies). Images were acquired with a Nikon A1 inverted confocal microscope, a Nikon Ti spinning disk confocal microscope (Nikon Instruments Inc.) and a Leica TCS SP8 microscope (Leica Biosystems).