Manual microtome

Histological slides were prepared by Saffron Scientific Histology Services (Carbondale, IL). Intestines previously fixed in 10% neutral buffered formalin were processed to paraffin using a Sakura enclosed automated tissue processor (Netherlands). The three representative areas of zebrafish intestines were orientated for cross sections embedded together in the same block. Five-micrometer serial sections were cut with a Leica manual microtome (Buffalo Grover, IL) and placed on water bath at 44°C. Sections were placed on positive charged slides. After drying, slides were stained with hematoxylin and eosin and cover-slipped using acrylic mounting media. The histological analysis focused on the hind-gut portions of fish digestive tract. Pictures of each slide at 100X magnification were obtained using a microscope (Leica DMI 300B) and camera (Leica DMC 290) combination, with the software LAS V4.4 (Leica Camera, Wetzler, Germany). From these pictures, individual lengths and widths were taken of intact villi using ImageJ (NIH, Betheseda, MD, USA). Length and width data were measured in the distal portion of the intestine. Villus length was measured from the tip of the villus to the luminal surface, and villi width was measured across the base of the villus at the luminal surface. The length-to-width ratio of each villus was determined by dividing the length by the width.

Lungs were fixed in 4% paraformaldehyde and processed then embedded in paraffin using an automated tissue processor (Leica, London, UK). Cross-sections (7 μm) were cut at 200 μm intervals throughout the lung using a manual microtome (Leica, London, UK). Contiguous sections were stained with haematoxylin and eosin (H&E), and for HIF1α, HIF2α, NIMP R14, Mac2, Annexin V, CD34, and proliferating cell nuclear antigen (PCNA) as described [9 (link),24 (link),25 (link),26 (link)].