Filter set 49

Cells (both non-induced and induced) were cultured in 6-well plates as described above. Prior to fluorescence microscopy imaging, both non-induced and induced cultures were stained with Vybrant® DyeCycle^TM Violet (2.5 μg/ml final concentration) and NR (final concentration 50 ng/ml). After a 20-minute incubation period (37 °C/5% CO₂), the culture medium was removed and the wells rinsed with PBS to remove non-adherent cells and unbound residual dye. Fluorescence images were captured using an AxioVert A1 inverted fluorescence microscope (Carl Zeiss, Gottingen, Germany) equipped with an AxioCam Cm1 camera (Carl Zeiss). Single channel images were captured and subsequently converted into overlay images. Nile Red was captured using Filter Set 9 (excitation BP 450–490, emission LP 515; Carl Zeiss) to visualise yellow-gold fluorescence and Vybrant® DyeCycle^TM Violet was captured using Filter Set 49 (excitation G 365, emission BP 445/50; Carl Zeiss) to visualise nuclei. Images were initially captured using AxioVision software (Version 4.8.2). In order to optimally visualise intracellular lipid droplets, all images were enhanced, but not manipulated, post-acquisition using Image J imaging software^{33 (link)}. Enhancement of images was done by adjusting contrast and brightness settings.

Tissue samples were fixed, sectioned, and either stained or labelled for light microscopy and photographed using a Carl Zeiss M2 AxioImager microscope (Betts et al., 2017b (link); Aubert et al., 2018 (link)). Sections stained with toluidine blue O (Sigma-Aldrich) were photographed using digital interference contrast (DIC). Calcofluor white- (Sigma-Aldrich) stained sections were viewed using Zeiss Filter set 49 (excitation 335–383 nm, emission 420–470 nm, exposure 230 ms; false-coloured turquoise). Immunofluorescence labelling of (1,3;1,4)-β-glucan and arabinoxylan was performed with the BG1 antibody (Meikle et al., 1994 (link)) and the LM11 antibody (McCartney et al., 2005 (link)), respectively, as described by Burton et al. (2011) (link), with the minor modifications outlined in Betts et al. (2017b) (link). Fluorescence labelling was observed using a Zeiss Fluorescence microscope (Axio Imager M2, Zeiss, Germany) with an AxioCam Mrm camera and processed using ZEN 2012 software (Zeiss, Australia).

Colorectal tissue was stored at −80 °C until processing. Cryosections of the colorectal tissue, containing the adenoma, were taken at 10 µm thickness and were mounted on indium tin oxide slides for MALDI MSI analysis. Serial sections were obtained for MALDI MSI and immunofluorescence microscopy using a pPDH antibody and DAPI staining. The cryosections used for immunofluorescence were 5 µm in thickness. Fluorescent microscopy images were acquired using a 40× objective (Zeiss Observer Z.1, Oberkochen, Germany), a DAPI filter (Filter Set 49, Carl Zeiss Microscopy, Oberkochen, Germany), and an FITC filter (31001, Chroma Technology Corporation, Bellows Falls, VT).