After deparaffinization, immunofluorescence (IF) staining was performed as described previously (17 (link), 21 (link)). Lung sections were stained with anti-HIF-1α antibody (Cat#: ab16066, Abcam) or anti- phospho-p65-NF-κB (pRelA) antibody (Cat#: 3033, Cell Signaling Tech). The slides were counter stained with anti-rabbit Alexa 564-conjugated secondary antibody (Cat#: A-11010, Life Technologies). Staining for CD31 (endothelial cell marker) was performed using rabbit anti-CD31 antibody (Cat#: ab28364; Abcam) at 1:2,000 dilution followed by DyLight 549–conjugated anti-rabbit antibody (Singh et al., 2013). HIF-1α-, p65-, and CD31-positive cells in the lungs were counted blind using Axiplan 2 imaging (Hamamatsu, Japan) on computer selected 9000 μm2 areas. Nuclei were stained with DAPI (blue fluorescence). The procedure was repeated three times with different animals.
Protocol detail
Immunofluorescence Staining of Lung Sections
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Animals
Anti antibody
Antibody
Blind
Cells
Dapi
Dilution
Endothelial cell
Fluorescence
Immunofluorescence
Lungs
Nf κb
Nuclei
Rabbit
Corresponding Organization : Lovelace Respiratory Research Institute
Other organizations : Florida International University, University of South Alabama, University of New Mexico, Johns Hopkins University
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Variable analysis
independent variables
- Anti-HIF-1α antibody (Cat#: ab16066, Abcam)
- Anti-phospho-p65-NF-κB (pRelA) antibody (Cat#: 3033, Cell Signaling Tech)
- HIF-1α-positive cells in the lungs
- P65-positive cells in the lungs
- CD31-positive cells in the lungs
- Lung sections
- Anti-rabbit Alexa 564-conjugated secondary antibody (Cat#: A-11010, Life Technologies)
- Rabbit anti-CD31 antibody (Cat#: ab28364; Abcam) at 1:2,000 dilution
- DyLight 549–conjugated anti-rabbit antibody
- DAPI (blue fluorescence)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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