Bx41 bright field light microscope

A BX41 bright field light microscope (Olympus) equipped with a DP72 digital camera (Olympus) and cellSens entry 1.4 software (Olympus) was used for bright field image acquisition. Fluorescence images were acquired with a spinning disk confocal microscope (Yokogawa) using Micro Manager acquisition software (v1.4.22). To prevent imaging of baseline lipofuscin autofluorescence, acquisition software settings were calibrated using a no-antibody negative control slide for each case. Images were acquired as a Z-series with a 1 μm step increment, with three or more high power fields imaged for each slide. Image processing was performed using free ImageJ software (v1.50i), with final editing done using Adobe Photoshop CC (2015.1.2 release).

Intestinal tissue was fixed with Carnoy’s fixative and embedded in paraffin, and sections were cut, then deparaffinized and rehydrated as above. The Merck-Millipore periodic acid–Schiff (PAS) staining kit (VWR) was used according to the manufacturer’s instructions to visualize goblet cells. In short, tissues were stained at room temperature with periodic acid solution for 10 minutes, Schiff’s reagent for 15 minutes, and Mayer’s hematoxylin solution (MilliporeSigma) for 30 seconds, with in-between washing steps with tap water and distilled water. After dehydration in an ascending alcohol series and xylene, sections were mounted with Entellan mountant (Merck) and analyzed using an Olympus BX41 Brightfield light microscope.