Neutrophil products and alterations in epithelial junctional proteins: prevention of artifactual degradation.
Recent reports of disruption of endothelial cell adherens junction proteins during neutrophil adhesion and transmigration have been challenged as being partly due to post-fixation artifactual release of neutrophil-derived proteases. In this study we examined alterations in the epithelial junctional complex during neutrophil adhesion. Using standard fixation protocols, neutrophil addition to epithelial monolayers resulted in gross disruption of apical junction protein immunofluorescence. However, the inclusion of a post fixation incubation step with formic acid resulted in epitope preservation. These observations indicate that neutrophil derived products, likely proteases, remain active despite prolonged exposure to conventional fixatives. This may result in diffuse and artifactual loss of epithelial junctional protein immunofluorescence. Formic acid prevents this loss of epitope staining and may be considered as an agent to preserve protease-sensitive endothelial or epithelial immunoreactivity.
Ginzberg
H
H
Department of Pediatrics, Division of Gastroenterology and Nutrition, The University of Toronto, Ontario, M5S 1A8, Toronto, Canada.
Shannon
P
P
Downey
G P
GP
D015820:Cadherins / Q000378:metabolism*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005561:Formates; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D015534:Trans-Activators; D014407:Tumor Cells, Cultured; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin
C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D005561:Formates; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; C030544:formic acid