SI and colonic GAPs were enumerated on fixed tissue sections as previously described7 (link),11 (link). Briefly, tetramethylrhodamine-labeled 10 kD dextran was administered in the jejunum and proximal colon of anesthetized mice. After 1 hour, mice were sacrificed and tissues thoroughly washed with cold PBS before fixing in 10% formalin buffered solution. Tissues were embedded in optimal cutting temperature compound (Fisher Scientific, Pittsburgh, PA); 7-μm sections were prepared, stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and imaged using an Axioskop 2 microscope (Carl Zeiss Microscopy, Thornwood, NY). GAPs were identified as dextran-filled columns measuring approximately 20 μm (height) × 5 μm (diameter) traversing the epithelium and containing a nucleus, and were enumerated as GAPs/villus cross section in the SI or GAPs/crypt cross section in the colon. Fixed SI tissue sections were stained with periodic acid-Schiff (PAS; Sigma-Aldrich) according to manufacturer’s instructions, and images were acquired on the Axioskop 2. In some experiments, mice were treated with 500 μg/kg tryphostin AG1478 (inhibitor of EGFR phosphorylation; Sigma-Aldrich) i.p. 30 min prior to luminal dextran administration.
Tryphostin ag1478
Manufactured by Merck Group
Tryphostin AG1478 is a specific inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. It functions by blocking the autophosphorylation of EGFR, thereby disrupting the EGFR signaling pathway.
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Lab products found in correlation
2 protocols using tryphostin ag1478
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Visualizing Intestinal Gap Junctions
2
Visualizing Intestinal Gap Junctions
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SI and colonic GAPs were enumerated on fixed tissue sections as previously described7 (link),11 (link). Briefly, tetramethylrhodamine-labeled 10 kD dextran was administered in the jejunum and proximal colon of anesthetized mice. After 1 hour, mice were sacrificed and tissues thoroughly washed with cold PBS before fixing in 10% formalin buffered solution. Tissues were embedded in optimal cutting temperature compound (Fisher Scientific, Pittsburgh, PA); 7-μm sections were prepared, stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and imaged using an Axioskop 2 microscope (Carl Zeiss Microscopy, Thornwood, NY). GAPs were identified as dextran-filled columns measuring approximately 20 μm (height) × 5 μm (diameter) traversing the epithelium and containing a nucleus, and were enumerated as GAPs/villus cross section in the SI or GAPs/crypt cross section in the colon. Fixed SI tissue sections were stained with periodic acid-Schiff (PAS; Sigma-Aldrich) according to manufacturer’s instructions, and images were acquired on the Axioskop 2. In some experiments, mice were treated with 500 μg/kg tryphostin AG1478 (inhibitor of EGFR phosphorylation; Sigma-Aldrich) i.p. 30 min prior to luminal dextran administration.
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