Brdu immunohistochemistry kit

The middle section of jejunum was obtained immediately after the mice were euthanized; the jejunum (about 10 cm in length) was rinsed with ice-cold PBS, and then it was fixed in 10% formalin for at least 24 h. H&E staining was performed following the above description. Relative height of villi was calculated by an Image-Pro Plus software (Version 6.1; Media Cybernetics). Analysis of apoptosis was performed using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (Roche, Basel, Switzerland) according to the manufacturer’s instructions. For 5-bromo-2ʹ-deoxyuridine (BrdU) staining, mice were injected with 100 mg/kg BrdU (Sigma, St. Louis, MO, USA) 2 h prior to sacrifice, sections from jejunum were deparaffinized and then a BrdU immuno-histochemistry kit was used according to the manufacturer’s instructions (Abcam, Cambridge, UK, USA) with modification, which epitope retrieved was executed 1 min by high pressure in a mix of citric acid and citrate solution. Apoptosis and proliferation were evaluated by TUNEL and BrdU-positive cells per crypt. All the images were captured using a Leica DMi8 fluorescence microscope (Leica, Solms, Germany).

Cell proliferation was evaluated by the BrdU-cell proliferation enzyme-linked immunoabsorbent assay (ELISA; Roche Applied Science, Mannheim, Germany) as described previously [31 (link),32 (link)]. The cells were seeded into 96-well culture plates at a density of 1×10⁴ cells/well. Cell proliferation was also evaluated visually under a BZ-9000 microscope (Keyence, Osaka, Japan) using the BrdU Immunohistochemistry Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.

AGS and HFE-145 cells were co-cultivated on glass cover slips in 6 well plates. After 45 hr co-cultivation, cells were incubated in media containing 10 µg/ml BrdU (Abcam, ab142567) for 3 hr. Cells were then fixed with 4% PFA for 15 min at RT. After washing 2 × with PBS, immunostaining of BrdU was performed using a BrdU immunohistochemistry kit (Abcam, ab125306) according to manufacturer’s protocol. Cover slips were then mounted onto slides using Prolong Diamond Antifade Mountant and left to dry for 24 hr. Slides were then imaged on a light microscope with 20 × objective and captured using an Olympus EP50 colour camera, with at least 10 separate locations imaged per repeat. BrdU-stained cells (brown) were counted and quantified as a percentage of the total cell population (counterstained blue with haematoxylin).