A lot of researchers commonly ask us if Visikol HISTO-M tissue clearing only helps when imaging 3D cell cultures (e.g. organoid, spheroid, microtissue) with confocal imaging. The answer to this question is “No.” While wide-field imaging will not allow for optical sectioning through tissues for spatial analysis, the addition of tissue clearing allows for a 3-4-fold increase in the number of cells that can be detected using wide-field imaging. The reason for this is that like confocal imaging, without tissue clearing, wide-field imaging is limited to characterizing the first few cell layers of 3D cell culture models. This limitation leads to the assumption that the peripheral cells in a 3D cell culture model are indicative of the entire population of cells which is typically not the case and can severely bias results.
Recently, we worked with Pandorum Technologies to image their hepatic organoid models using Visikol HISTO-M on a Thermo Fisher EVOS FL Auto II. Pandorum was able to demonstrate that Visikol HISTO-M dramatically improved image quality while being compatible with antibody labeling and nuclear staining. Models were labeled for Albumin (cat no. MAB1455- R&D System), BSEP (cat no. sc74500- Santacruz), ZO-1 (cat no. 61-7300- Thermo Fisher Scientific), E-cadherin (cat no. sc71009- Santacruz), Vimentin (cat no. sc6260- Santacruz) and Goat anti-mouse Alexa Fluor 488 secondaries (cat no. A-11029 Thermo Fisher).
Treatment with Visikol HISTO-M occurs after labeling and is compatible with traditional tissue processing and labeling workflows. We provide protocol guidance and labeling optimization guidance through our Protocol Guidebook.
Hepatic organoids were made in 1ul starting volume with 10000 cells. The organoids were matured for 7 days and then stained in the 96 well plate. The EVOS FL auto II microscope has the option of capturing the complete well of a 96 well plate in multi-channel and in Z stacks and digitally stitching the complete well. We performed this function for the organoids stained for albumin, BSEP and E-cadherin- treated with Visikol (bottom panel) and without (top panel), scale bar 450 µm