Endobronchial biopsies were performed using disposable biopsy forceps with jaws closed at the interbronchial carina before retracting. Tissue was fixed immediately in 4% paraformaldehyde and paraffin embedded. CD4+ T cells were identified by staining with mouse anti-CD4 (Dako) at 1:100 dilution using the EnVision peroxidase staining method (Dako) as previously described (62 (link)). Slides were coded to avoid observer bias and assessed using a Leitz Dialux 20 light microscope and Image 1.5 software. Total epithelial and subepithelial areas of 2 to 3 bronchial biopsies were counted at each time point. Cell counts were expressed as the number of cut cell profiles with visible nucleus per mm2 of subepithelium and per 0.1 mm2 of epithelium. The coefficient of variation for repeat counts of positive cells by a single observer ranged from 5% to 6%.
Envision peroxidase staining method
Manufactured by Agilent Technologies
Sourced in Denmark
The EnVision peroxidase staining method is a laboratory technique used to detect and visualize specific target molecules, such as proteins, in tissue samples. It employs a peroxidase-based detection system to amplify the signal and enhance the visibility of the target analyte. The core function of this method is to provide a sensitive and reliable way to identify and localize target molecules within a sample, enabling researchers to study their distribution and expression patterns.
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3 protocols using envision peroxidase staining method
1
Endobronchial Biopsy Cell Quantification
2
Quantification of RSV and CD8+ T Cells
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Endobronchial biopsies were fixed immediately in 4% paraformaldehyde and paraffin embedded. RSV was stained using polyvalent mouse anti-RSV antibody (NCL-RSV3, Leica Biosystems, UK) at 1:50 dilution. CD8+ T cells were identified by staining with mouse anti-CD8 (M0707, Dako) at 1:100 dilution using the EnVision peroxidase staining method (Dako, Denmark) as previously described30 (link). Briefly, 5-μm sections were stained according to the manufacturer's instructions and an irrelevant mouse IgG1 kappa antibody (MOPC21) was used as negative control for staining specificity of mouse monoclonal antibodies. RSV-infected A549 cells were used as positive staining controls. Quantification was achieved as previously described with operators blinded to sample timing and infection status30 (link). Briefly, slides were coded to avoid observer bias and areas of epithelium and subepithelium assessed using a Leitz Dialux 20 light microscope, Apple Macintosh computer and Image 1.5 software. Total epithelial and subepithelial areas of two to three bronchial biopsies were counted for each bronchoscopy. Cell counts were expressed as the number of cut cell profiles with visible nucleus per mm2 of subepithelium and per 0.1 mm2 of epithlium. The coefficient of variation for repeat counts of positive cells by a single observer ranged from 5 to 6%.
3
Quantifying RSV and CD8+ T Cells in Endobronchial Biopsies
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