Axioimager system

Paraffin-embedded tissue sections (5 μm thick) of small airways and alveoli were deparaffinized and hydrated in order to conduct immunofluorescent histochemical analyses. The sections were preincubated in a boiling sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for the antigen retrieval. The tissues were blocked with 5% BSA in PBS for 1 h. A specific primary antibody against MMP-12 was incubated overnight with the sectioned tissues. Subsequently, the tissue sections were incubated for 1 h with fluorescein isothiocyanate-conjugated or Cy3-conjugated anti-rabbit IgG. For identification of nuclei, the fluorescent nucleic acid dye of DAPI was applied for 10 min. Stained tissues were mounted on slides using mounting medium (Vector Laboratories, Burlingame, CA, USA). Images of each slide were obtained with an optical microscope Axioimager system (Zeiss).

For the histological analyses of airways, small airway and alveolar specimens provided at the end of the experiments were fixed in 10% paraformaldehyde. The paraffin-embedded specimens were sectioned at 5 μm thickness, deparaffinized and stained with H&E stain for 2 min, and quickly dehydrated in 95% absolute alcohol. The H&E-stained tissue sections were examined using an optical microscope Axioimager system equipped for fluorescence illumination (Carl Zeiss). Five images were taken from each tissue section.

Tissue samples from the lung were collected and fixed in 10% neutral buffered formalin, which was further processed in an automated tissue processor and embedded in paraffin wax before triplicate sections were prepared. Paraffin-embedded tissue sections (5 μm thickness) of small airways and alveoli were deparaffinized and hydrated to conduct immunofluorescent histochemical analyses. The sections were preincubated in a boiling sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for antigen retrieval. The tissues were blocked with 5% BSA in phosphate-buffered saline for 1 h. A specific primary antibody against TF, CD11b or F4/80, was incubated overnight with the sectioned tissues. Subsequently, the tissue sections were incubated for 1 h with Cy3-conjugated anti-rabbit IgG or FITC-conjugated anti-rat IgG. For identification of nuclei, the fluorescent nucleic acid dye of DAPI was applied for 10 min. Stained tissues were mounted on slides using a mounting medium (Vector Laboratories, Burlingame, CA, USA). Images of each slide were obtained with an optical microscope Axioimager system equipped for fluorescence illumination (Zeiss, Gottingen, Germany).

Paraffin-embedded tissue sections (5 μm thick) of alveoli were deparaffinized and hydrated in order to conduct immunofluorescent histochemical analyses. The sections were preincubated in a boiling sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for the antigen retrieval. The tissues were blocked with 5% BSA in PBS for 1 h. A specific primary antibody against CD68 was incubated overnight with the sectioned tissues. Subsequently, the tissue sections were incubated for 1 h with fluorescein isothiocyanate-conjugated anti-rabbit IgG. For identification of nuclei, the fluorescent nucleic acid dye of DAPI was applied for 10 min. Stained tissues were mounted on slides using mounting medium (Vector Laboratories, Burlingame, CA, USA). Images of each slide were obtained with an optical microscope Axioimager system (Carl Zeiss).

For the immunohistochemical analysis, paraffin-embedded kidney tissue sections (3 μm thick) were employed. The sections were placed on glass slides, de-paraffinated and hydrated with xylene and graded alcohol. The sections were pre-incubated in a boiling sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for antigen retrieval. Specific primary antibody against mouse α-SMA and AGE was incubated with the tissue sections overnight. For the α-SMA localization, the sections were stained with FITC-conjugated anti-rabbit IgG. Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI). For the AGE visualization, the sections were developed with 3,3′-diaminobenzidine (DAB) as a substrate to produce brownish staining and counter-stained with hematoxylin. Each slide was mounted in VectaMount mounting medium (Vector Laboratories, Burlingame, CA, USA). The stained tissue sections were taken with an optical microscope Axioimager system (Zeiss, Oberkochen, Germany) and images and their intensity were obtained for each section (Auto-measure Axio Vision program).