Az100m widefield microscope

For imaging of IHC, EdU, or TUNEL staining, a META Zeiss LSM Meta 510 confocal microscope was used through the Vanderbilt Center for Imaging Shared Resource (CISR) Core. Slides stained with SA-βGal and ToPro were imaged using a Nikon AZ100M Widefield microscope through the Vanderbilt CISR Core. Confocal images were processed using Zen Blue version 3.1 with follow up analyses done using ImageJ. AZ100M images were first processed using NIS-Elements Viewer 5.21 and then subsequently analyzed using ImageJ.

For cryosectioning, thymi were snap-frozen on dry ice and stored at −80°C. Tissues were sectioned at 8-μm thickness and fixed in ice-cold acetone for 5 min. Tissues were rinsed with PBS, blocked with 10% donkey serum in PBS for 30 min at room temperature, and incubated with the primary antibody anti-Keratin 5 (Abcam) or biotin-labeled UEA-1 lectin (Vector Labs) overnight at 4°C. Secondary detection was performed with goat anti-rabbit IgG (Jackson ImmunoResearch) and Streptavidin-Cy3 (Vector Labs). Sections were examined by fluorescent microscopy using a Zeiss LSM 710 confocal microscope.
For paraffin sectioning, tissues were collected and fixed in 10% paraformaldehyde for formalin overnight. Tissues were dehydrated through an ethanol series and embedded in paraffin wax using standard procedures. Sections (8 μm) were cut and rinsed in xylene before rehydration through a reverse ethanol series. H&E staining was performed on paraffin sections using standard procedures; sections were then imaged on a Nikon AZ 100M wide-field microscope.