Thunder 3d live cell imaging system

Deparaffinized tissue sections were incubated with hydrogen peroxide for 5 min to block endogenous peroxidase, then incubated for 30 min in blocking buffer (5% BSA, 5% heat-inactivated normal goat serum in PBST) for 30 min. After overnight incubation with primary anti-Lyz antibody (Dako), sections were repeatedly washed in PBST and incubated with biotinylated secondary antibody for 1 h at RT and then visualized with VECTASTAIN ABC HRP Kit (Vector Laboratories) as indicated by the manufacturer. Sections were then incubated in a 3% aqueous solution of acetic acid for 3 min and then mucins were stained with alcian blue (1 g in 100 mL 3% acetic acid) for 15 min. Hematoxylin (50% aqueous solution) staining was then carried out according to standard procedure. Sections were imaged on Leica THUNDER 3D Live Cell Imaging system.

Mice were sacrificed and the duodenum was removed and flushed thoroughly in ice-cold PBS. Duodenal tissue was then cut in 1 cm pieces and fixed in 4% PFA (Santa Cruz Biotechnology, sc-281692) in PBS for 3 h and subsequently agitated in 30% sucrose, 4% PFA in PBS overnight at 4 °C. Fixed tissues were embedded in TIssue-Tek OCT Compound (Sakura, 4583). Eight micrometers thick sections were sectioned onto poly L-lysine coated coverslips. Probe libraries were designed using the Stellaris FISH Probe Designer Software (Biosearch Technologies) (see Supplementary Data 1) and coupled to Cy5 (Myc, Fos, Creb3l3) or TMR (Lgr5, JunB) as described^{48 (link)}. The intestinal sections were hybridized with smFISH probe sets according to a previously published protocol⁴⁹ . DAPI (Sigma-Aldrich) was used as a nuclear counterstain. smFISH imaging was performed on a Leica THUNDER 3D Live Cell Imaging system, using the following THUNDER Computational Clearing Settings in with the LAS-X software: Feature Scale (nm): 350, Strength (%): 98, Deconvolution settings: Auto and Optimization: High. The of Lgr5+ area per crypt was manually quantified using FIJI. We quantified at least 8 crypts from 2 to 3 different mice per condition. Barplots were generated on GraphPad Prism. The unpaired Student’s T test function in GraphPad Prism was used to analyze the significance of two-group comparisons.

The ABEmax probe library was designed using Stellaris FISH Probe Designer Software (Biosearch Technologies) (Supplementary Table 7) and coupled to Cy5 as described⁵⁹ . Livers were fixed in 4% PFA in PBS for 3 h and subsequently agitated in 30% sucrose and 4% PFA in PBS overnight at 4 °C. Fixed tissues were embedded in Tissue-Tek OCT Compound (Sakura, 4583). Then 8-μm-thick sections were sectioned onto poly-l-lysine-coated coverslips, air dried for 5 min, fixed for 15 min in 4% PFA and permeabilized overnight in 70% EtOH. The liver sections were hybridized with single-molecule fluorescence in situ hybridization (smFISH) probe sets according to a previously published protocol⁶⁰ . DAPI (Sigma-Aldrich) was used as nuclear counterstain. smFISH imaging was performed on a Leica THUNDER 3D live cell imaging system, using the following THUNDER computational clearing settings: Feature Scale (nm): 350; Strength (%): 98; Deconvolution settings: Auto; and Optimization: High.