ATII cells or paraffin-embedded human lung tissue sections were incubated with SP-C, TOP1-cc (both from Millipore), SP-A (Novus Biologicals, Littleton, CO, USA), pro-SP-C, p-DRP1, TOM20, MFN1, OPA1, FIS1, p63, CD68, TDP1 or DJ-1 (all from Santa Cruz Biotechnology). Secondary antibodies Alexa Fluor 594, Alexa Fluor 488 or Alexa 647 (Invitrogen Corp., Carlsbad, CA, USA) were applied for 1 h. Mitochondrial nucleoids were identified by DNA-intercalating dye Picogreen (Lumiprobe, Hunt Valley, MD, USA) as previously described [29 (link),31 (link)]. Sections were mounted with Vectashield medium containing DAPI (Abcam) to detect nuclei. Images were obtained using a confocal laser-scanning microscope (Zeiss). Pearson's correlation coefficient was used to analyze the colocalization of proteins of interest and TOM20 in SP-A-positive ATII cells in non-smokers, smokers, and patients with emphysema. Protein fluorescence intensity and colocalization were quantified by Image J (NIH) and normalized to control non-smokers as 1.
Vectashield medium
Manufactured by Abcam
Vectashield medium is a mounting medium that is commonly used in fluorescence microscopy applications. It is designed to protect fluorescent signals from photobleaching and maintain the optical clarity of mounted samples.
Automatically generated - may contain errors
Lab products found in correlation
Tom20, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
A confocal laser scanning microscope, by Zeiss (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Pro spc, by Santa Cruz Biotechnology (1 mentions)
Top1 cc, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594 igg, by Thermo Fisher Scientific (1 mentions)
Cys106 oxidized dj 1 hca024, by Bio-Rad (1 mentions)
Alexa fluor 647, by Novus Biologicals (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
2 protocols using vectashield medium
1
Immunofluorescence Analysis of Lung Tissue
2
Localization of Oxidized DJ-1 in Lung Cells
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Localization of oxidized DJ-1 form was analyzed in cultured A549 cells treated with 1 mM H2O2 as described above. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 3% normal donkey serum (Jackson ImmunoResearch, West Grove, PA). Cells were incubated overnight with Cys106-oxidized DJ-1 (HCA024, Bio-Rad, Hercules, CA) and Tom20 (Santa Cruz Biotechnology, Dallas, TX) antibodies. The secondary antibodies, Alexa Fluor 488 and Alexa Fluor 594 IgG (Invitrogen, Carlsbad, CA) were applied for 1 h. The cells were then mounted with Vectashield medium containing DAPI (Abcam, Cambridge, MA) and analyzed using a fluorescence microscope (Olympus). We also analyzed DJ-1 oxidation in ATII cells using lung tissue obtained from non-smokers, smokers and emphysema patients. Sections were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), embedded in paraffin, deparaffinized and hydrated followed by antigen retrieval as we previously described27 (link). SP-A (Novus, Biologicals, Littleton, CO) was used to identify ATII cells followed by incubation with Alexa Fluor 647 as described above.
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