Electronic supplemental material- appendix 1
Histology. The six branches dedicated for histological analyses (t = 24 weeks only) were decalcified in 5% EDTA (in phosphate buffered saline [PBS], pH 7.4) on ice for ~7 d. Both the EDTA solution and ice were replenished every 12 h. Once only the tissue tunics remained, they were washed thrice with PBS and then dehydrated in 70% ethanol for 30 min. Successive dehydrations at 80, 90, 99.9, and 99.9% ethanol for 30, 30, 60, and 60 min, respectively, were then performed. Tissues were then cleared twice with xylene for 30 min and embedded with 1:1 xylene:paraffin (Paraplast®, Leica, Buffalo Grove, IL, USA) for 20 min in a vacuum oven (DOV-30, Deng Yng Co., Ltd., Taipei, Taiwan) at 5 kPa and 60°C. Then, tissues were embedded twice with 100% paraffin at 5 kPa and 60°C, transferred to tissue molds, and overlaid with molten paraffin with a Thermo-Shandon Histocentre 2 histological station (Kalamazoo, MI, USA). Samples were allowed to harden overnight at room temperature (RT) and then on ice for 1 h prior to removing the tissue cassette from the mold.
Six micron sections made with a Thermo-Shandon Finesse 325 microtome were expanded in a 50°C water for 10 s. Then, the tissue sections were placed on glass microscope slides and allowed to dry at 50°C for 1 h. Tissue sections were stained with hematoxylin and eosin, coated with Histomount (National Diagnostics, Cherry Hill, NJ, USA) and a cover slip, and visualized under a light microscope (Olympus C31, Tokyo, Japan) at 200 or 400x. The thickness of the epiderm and the gastroderm was calculated with Micrometrics SE3 (Taipei, Taiwan) software in 10 randomly chosen locations within one tissue section from each of 10 randomly chosen polyps in each of the six samples. The average epiderm/gastroderm thickness ratio was then calculated from the 100 data points for each sample (n = 3 biological replicates from each treatment).
SEM. For the six branches dedicated for SEM (t = 36 weeks only), decalcification was accomplished with incubation in 10% formic acid for 6-12 h. Then, the tissue tunics were washed thrice with PBS for 10 min each wash and post-fixed in 1% osmium tetroxide in PBS for 1 hr at RT. Osmium tetroxide was removed with three washes with PBS. Then, tissues were incubated in 25% DMSO in PBS for 30 min, followed by 50% DMSO in PBS for 30 min. Afterwards, permeabilized tissues were immersed in liquid nitrogen for 1-2 min to freeze-fracture them. This allows for easier visualization of the epiderm-gastroderm interface. After the tissues were completely frozen, they were crushed with tweezers and re-immersed in 50% DMSO in PBS for 30 min. Then, tissues were washed thrice in PBS for 10 min each and dehydrated as described above for histology except with an initial step in 50% ethanol for 20 min.
To fully dehydrate the tissues, a critical point dryer (HCP-2, Hitachi, Tokyo, Japan) was used according to the manufacturer’s recommendations. Fully dehydrated tissues were coated with platinum for 40 s in an Ion Sputter (E1010, Hitachi) according to the manufacturer’s recommendations and visualized with a S-3500N scanning electron microscope (Hitachi) as in Tsai et al. (2010). Image J (National Institutes of Health, Bethesda, MD, USA) was used to measure epiderm and gastroderm tissue thickness in 10 randomly selected portions of each of 10 micrographs (each from a different polyp) from each sample, and an average was calculated across the 100 data points for each sample (n = 3 biological replicates from each treatment). The thickness of the individual layers, the total thickness of both layers, and the epiderm/gastroderm tissue thickness ratio were all analyzed as described in the text (“Statistical analyses”).