Provis ax70 widefield microscope

The small intestine was isolated, washed in PBS and cut into three segments, namely duodenum, jejunum and ileum, to fix for 24 h in 10% formaldehyde. Paraffin sections from fixed segments were cut into 10 μm sections and prepared to be stained with Haematoxylin and Eosin (H&E). Briefly, sections were immersed in acidified Haematoxylin for 1 min, and rinsed with tap water. The sections were stained using Eosin for 1–2 mins followed by washing. Ascending alcohol solutions (50%, 70%, 80%, 95% x 2, and 100% x 2) were applied to dehydrate tissue sections, and cleared using xylene. Sections were viewed using an Olympus Provis AX70 widefield microscope and histological quantification of neutrophil number per segment was performed using at least five transverse sections per mouse in a blinded procedure.

Sections were prefixed with 4% PFA in PB for one hour in a humid chamber at room temperature, then washed twice for 15 minutes with 0.3% Triton X-100 in 1x PBS (PBS-Tx (0.3%)). The pH of sections was then equilibrated with two 30 minute washes with 1x PBS at pH 5.5 (all washes were performed in a humid chamber). Sections were then stained for 16 hours at 37°C in a humid chamber in staining solution (2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K3Fe(CN)6·3H2O, 1 mg/ml X-gal, with the remaining volume made up of 1x PBS at pH 5.5). Following incubation, staining was arrested by a 20-minute wash in 4% PFA in PB at room temperature. Sections were then washed well in PBS at pH 5.5 and mounted in 50% glycerol. Sections were then imaged on an Olympus Provis AX70 widefield microscope with an Olympus DP70 camera, with images acquired at 4080x3072 resolution.

Total follicle numbers (oocyte present) were counted in every ninth resin section using the Provis AX70 Widefield Microscope (Olympus). Cross-sectional tissue area was measured using CellSens Software (Olympus). Follicle density (n = 4–5/age/treatment/time point) was reported as average number of follicles per area (mm²).