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The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice

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Abstract

Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr−/− mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.

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Abbreviations

AB-PAS:

Alcian Blue–periodic acid Schiff

AEBSF:

4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride

BSA:

Bovine serum albumin

BCA:

Bicinchoninic acid

CFTR:

Cystic fibrosis transmembrane conductance regulator

DIG:

Digoxigenin

IHC:

Immunohistochemistry

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde

RT:

Room temperature

SSC:

Saline sodium citrate

TCA:

Trichloroacetic acid

vWF:

von Willebrand factor

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Acknowledgments

We thank Delphine Fontaine (Inserm U837) for her excellent technical assistance in performing IHC, the Microbiology Department of the “Centre de Biologie-Pathologie Pierre-Marie Degand” (Lille Hospital) for the access to the ABI Prism 7700, MH Gevaert and RM Siminski (Service commun–Morphologie cellulaire, Université Lille 2) for slides and J. Bara (Inserm UMR S938, Paris, France) for the 45M1 antibody. This study was supported by the French Cystic Fibrosis association “Vaincre la Mucoviscidose” (J.L.D., FC0203 and IC0806) and Children’s Memorial Research Center (S.H.L. and A.H.).

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Correspondence to Jean-Luc Desseyn.

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Gouyer, V., Leir, SH., Tetaert, D. et al. The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice. Histochem Cell Biol 133, 517–525 (2010). https://doi.org/10.1007/s00418-010-0688-8

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