Novel use of confocal microscopy in conjunction with EdU technology to determine in vivo cellular proliferation in the piglet intestine

Abstract

The method most commonly used to label newly divided cells is a thymidine analogue, 5‐bromo‐2′‐deoxyuridine (BrdU). BrdU detection requires extensive DNA denaturation. However, an alternative thymidine analogue, 5‐ethynyl‐2′‐deoxyuridine (EdU) can be used. EdU that has been incorporated into newly formed DNA is tagged by a covalently bound fluorescent azide molecule allowing for highly specific recognition without DNA disruption. We have optimized EdU technology for in vivo use in pigs using confocal microscopy as a novel approach to quantify proliferation in ex vivo stained sections. Piglets were intraperitoneally injected with 10mM EdU at 1 ml per kg body weight 2 hours prior to euthanasia on day 7 or 14 postpartum. Formalin‐fixed jejunum and ileum sections were stained using the Click‐iTTM EdU Alexa Fluor 555 Imaging Kit (Invitrogen). DAPI was used to detect total nuclei. Samples were imaged using Confocal and Nanozoomer microscopy. AxioVision was used to quantify the area of EdU per unit area of DAPI. We have shown the use of confocal microscopy provides better specificity and depth discrimination than Nanozoomer microscopy. This is necessary to accurately detect small changes in proliferation from thick tissue specimens. These findings will be used in future studies to detect changes in intestinal proliferation caused by feeding of bioactive compounds to neonatal piglets.