Immunohistochemical Localization of Bacteria

Infected BBEC cultures representing each time point p.i. were fixed, processed, and sectioned as previously described (58 (link)), and the sections were subjected to either H&E staining or IHC staining. In the latter case, bacteria were identified by incubation for 30 min with a 1:800 dilution of rabbit anti-OmpA antibody (103 (link)), application of an anti-rabbit horseradish peroxidase (HRP)-labeled polymer, and visualization with a Real EnVision peroxidase/DAB⁺ detection system (Dako; catalog no. K3468); samples were subsequently counterstained with Gill’s hematoxylin. Tissue sections were viewed with a Leica DM2000 light microscope.

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Cozens D., Sutherland E., Lauder M., Taylor G., Berry C.C, & Davies R.L. (2019). Pathogenic Mannheimia haemolytica Invades Differentiated Bovine Airway Epithelial Cells. Infection and Immunity, 87(6), e00078-19.

Publication 2019

Real EnVision peroxidase/DAB+ detection system DM2000 light microscope

Anti antibody Bacteria Dilution Gill Hematoxylin Horseradish peroxidase Microscope light Peroxidase Polymer Rabbit Tissue

Corresponding Organization :

Other organizations : University of Glasgow, The Pirbright Institute

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Variable analysis

independent variables

Time points post-infection (p.i.)

dependent variables

Bacterial presence and localization as determined by immunohistochemical (IHC) staining using anti-OmpA antibody

control variables

BBEC (bronchial epithelial cell) cultures
H&E staining method
Rabbit anti-OmpA antibody (1:800 dilution)
Anti-rabbit horseradish peroxidase (HRP)-labeled polymer
Real EnVision peroxidase/DAB+ detection system
Gill's hematoxylin counterstain
Leica DM2000 light microscope

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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