Dapi dihydrochloride

Formalin-fixed paraffin-embedded intestinal tissue sections (5 μm) were initially treated with lysis buffer for 1 h at 37°C and then hybridized at 51°C with a 20 bp bacteria-specific probe (EUB 338: GCT-GCCTCCCGTAGGAGT) to visualize and quantify the proximity of luminal bacterial colonies and the corresponding antigen load on the epithelium. Following the overnight hybridization, the intestinal sections were counter-stained with 4,6-diamidino-2-phenylindole (DAPI, Dihydrochloride, Cat #: D1306 by Invitrogen) for visualization of cell nuclei. Images were obtained and analyzed using a Keyence BZ-X810 all in one fluorescence microscope.

Immunofluorescent staining and imaging strategies for half-scaffold constructs have been previously reported.¹⁷ Briefly, seeded silk scaffolds with crypt and villi architecture were fixed and subsequently trimmed to expose the crypts and villi to the blocking buffers and antibodies. Following permeabilization and blocking, the scaffolds were incubated overnight (12–16 h) at 4 °C with anti-human ZO-1 conjugated with Alexa Fluor 594 Conjugate (Thermofisher, 1:100), or primary antibodies including anti-human-MUC-2 (Santa Cruz Biotech, 1:50) and anti-Sucrose isomaltase (abcam, 1:100). The following day, the primary antibodies were removed from the scaffolds. The scaffolds were then stained with Alexa Fluor 488 donkey anti-mouse secondary antibody at 1:250 (Invitrogen) and counterstained with dihydrochloride (DAPI, Invitrogen) for nuclei. The stained specimens were mounted glycerin, and the luminal surface of the scaffolds was observed and laser-scanned under a Leica SP8 confocal microscope with a filter set for DAPI (Ex/Em: 350/470 nm), Texas Red (Ex/Em: 540/605 nm) and GFP/FITC (Ex/Em: 488/514 nm). Leica confocal software (application suite X, 3.3.0.16799 and ImageJ) was used to create 3D rendered images.