The study was reviewed and approved by the Duke University Institutional Review Board (Durham, NC). An existing cohort of patients diagnosed to have chronic obstructive pulmonary disease (COPD; some combination of chronic bronchitis and emphysema) was searched and six individuals identified. Blocks of lung tissue collected at autopsy were retrieved from archives.
Perls’ Prussian blue was employed to stain iron. Hale’s stain was used as an assay for in situ iron binding capacity20 (link). The background stain was nuclear fast red. Tissue was stained for an iron importer and storage protein. Five micron tissue sections were cut, floated on a protein-free water bath, mounted on silane treated slides, and air-dried overnight. Sections were then deparaffinized and hydrated to 95% alcohol (xylene for 10 min, absolute alcohol for 5 min, and 95% alcohol for 5 min). Endogenous peroxidase activity was blocked with 0.6% H2O2 in absolute methanol for eight minutes. Slides were rinsed in 95% alcohol for 2 min, placed in deionized H2O, and washed in PBS. After treatment with Cyto Q Background Buster (Innovex Biosciences, Richmond, CA) for 10 min, slides were incubated with the primary antibody diluted in 1% bovine serum albumin for 45 min at 37 °C in PBS. Primary antibodies used in this investigation were to divalent metal transport 1 (DMT1) (generously provided by Dr. Funmei Yang of the University of Texas, San Antonio, TX) used at a dilution of 1:200 and ferritin (Dako, Carpinteria, CA) used at a dilution of 1:200. Slides were incubated with biotinylated linking antibody from Stat-Q Staining System (Innovex Biosciences) for ten minutes at room temperature, washed with PBS, and peroxidase enzyme label from Stat-Q Staining System (Innovex Biosciences) applied. After incubation for ten minutes at room temperature and washes with PBS, tissue sections were developed with 3,3′diaminobenzidine-tetrahydrochloride for three minutes at room temperature. Sections were counterstained with hematoxylin, dehydrated through alcohols, cleared in xylene and coverslipped using a permanent mounting media. Photomicrographs were obtained using a Nikon Eclipse E600 microscope (Tokyo, Japan) with 10×/40× objective lens coupled with QCapture software (QImaging, Surrey, British Columbia, Canada).
Perls’ Prussian blue was employed to stain iron. Hale’s stain was used as an assay for in situ iron binding capacity20 (link). The background stain was nuclear fast red. Tissue was stained for an iron importer and storage protein. Five micron tissue sections were cut, floated on a protein-free water bath, mounted on silane treated slides, and air-dried overnight. Sections were then deparaffinized and hydrated to 95% alcohol (xylene for 10 min, absolute alcohol for 5 min, and 95% alcohol for 5 min). Endogenous peroxidase activity was blocked with 0.6% H2O2 in absolute methanol for eight minutes. Slides were rinsed in 95% alcohol for 2 min, placed in deionized H2O, and washed in PBS. After treatment with Cyto Q Background Buster (Innovex Biosciences, Richmond, CA) for 10 min, slides were incubated with the primary antibody diluted in 1% bovine serum albumin for 45 min at 37 °C in PBS. Primary antibodies used in this investigation were to divalent metal transport 1 (DMT1) (generously provided by Dr. Funmei Yang of the University of Texas, San Antonio, TX) used at a dilution of 1:200 and ferritin (Dako, Carpinteria, CA) used at a dilution of 1:200. Slides were incubated with biotinylated linking antibody from Stat-Q Staining System (Innovex Biosciences) for ten minutes at room temperature, washed with PBS, and peroxidase enzyme label from Stat-Q Staining System (Innovex Biosciences) applied. After incubation for ten minutes at room temperature and washes with PBS, tissue sections were developed with 3,3′diaminobenzidine-tetrahydrochloride for three minutes at room temperature. Sections were counterstained with hematoxylin, dehydrated through alcohols, cleared in xylene and coverslipped using a permanent mounting media. Photomicrographs were obtained using a Nikon Eclipse E600 microscope (Tokyo, Japan) with 10×/40× objective lens coupled with QCapture software (QImaging, Surrey, British Columbia, Canada).
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