Observer d1 inverted phase contrast fluorescence microscope

Five micrometer thick paraffin-embedded tissue sections were deparaffinized and rehydrated using Xylene and a descending ethanol gradient (100%, 95%, 70%, pure dH₂O). Three percent acetic acid was applied to the slides for 3 min. Alcian blue solution (pH 2.5) was added to slides for 30 min to stain the goblet cells. Three percent acetic acid was applied to the slides for ~30 s to remove excess Alcian blue staining. Slides were rinsed in running tap water for 5 min followed by two changes of distilled water. Nuclear Fast Red solution was applied for 5 min to stain nuclei. Slides were rinsed with running tap water for 2 min, transferred to two changes of distilled water, and then dehydrated with an ascending ethanol gradient (70, 95, and 100%). Slides were mounted. Images were acquired using a Zeiss Observer D1 Inverted Phase Contrast Fluorescence Microscope, Axiocam 105 color camera, and Zeiss Zen pro microscope software (Stony Brook University).

Five micrometer thick paraffin-embedded tissue sections were deparaffinized and rehydrated using Xylene and a descending ethanol gradient (100%, 95%, 70%, pure dH₂O). Mayer’s Haematoxylin solution was used to stain nuclei to a medium density (~1 min). Slides were thoroughly rinsed in running tap water for 5 min and stained with phloxine solution for 20 min. Slides were rinsed with tap water, blotted dry, and rinsed with tartrazine solution to remove remaining water. Slides were kept in tartrazine solution and visualized under a microscope until granules were red and all other tissue was yellow. Slides were dehydrated in 70, 95, and 100% ethanol and then placed in two washes of 100% xylene before being mounted. For analysis of average Paneth cell number per crypt, at least 10 crypts were counted per mouse. Images were acquired using a Zeiss Observer D1 Inverted Phase Contrast Fluorescence Microscope, Axiocam 105 color camera, and Zeiss Zen pro microscope software (Stony Brook University).