C1 laser scanning confocal microscope system

Paraffin-embedded sections were deparaffinized, rehydrated, then steamed for 30 min in citrate buffer for antigen retrieval, after blocking 5% goat serum in Tris-buffered saline with 0.05% Tween 20. Sections were incubated with primary antibodies against either Ki-67 (1:100, ThermoFisher Scientific, Waltham, MA, USA) or Phospho-S6 Ribosomal Protein (1:400, Cell Signaling Technology). Horse-radish peroxidase-conjugated secondary antibodies (rabbit anti-goat IgG 1:200, Abcam, Cambridge, MA, USA, and goat anti-rabbit IgG, 1:100: Abcam) followed by the SignalStain DAB Substrate Kit (Cell Signaling Technology) were used for detection. Nuclear counterstaining was performed using hematoxylin. For apoptosis detection, ileal sections were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using the In Situ Cell Death Detection Kit, TMR red (Roche Diagnostics, Indianapolis, IN, USA), per manufacturer’s instructions. Sections were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI (4′,6-diamidino-2-phenylindole) as a nuclear stain. Slides were visualized with the C1 laser scanning Confocal Microscope System (Nikon, Melville, NY, USA), and analyzed using ImageJ.

Paraffin-embedded sections (5-μm thick) were deparaffinized, rehydrated, then steamed for 30 min in citrate buffer for antigen retrieval. Sections were blocked with 5% goat serum in Tris-buffered saline with 0.05% Tween 20, incubated with a primary antibody for Ki-67 (1:100, ThermoFisher Scientific, Waltham, MA, USA) overnight, followed by Alexa Fluor 647-conjugated secondary antibody. For detection of apoptosis, ileal sections were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using the In-Situ Cell Death Detection Kit, TMR red (Roche Diagnostics, Indianapolis, IN, USA), as per the manufacturer’s instructions. All sections were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI (4′,6-diamidino-2-phenylindole) as a nuclear stain. Slides were visualized with the C1 laser scanning Confocal Microscope System (Nikon, Melville, NY, USA).