4.6. Immunohistochemistry for α-SMA and Desmin

Sections of lung (4 μm thick) were cut from each paraffin block and then deparaffinized with xylol and ethanol. After rinsing with phosphate-buffered saline (PBS), sections were incubated overnight at 4 °C with anti-rat-α-SMA monoclonal antibody (1:100 dilution; Dako, Carpinteria, CA, USA) and anti-rat-desmin polyclonal antibody (1:500 dilution; Abcam, Cambridge, MA, USA). Sections were then rinsed in PBS and incubated for 90 min with Alexa^® Fluor 488-labeled rabbit anti-mouse Immunoglobulin G (IgG) antibody and Alexa Fluor 594-labeled mouse anti-rabbit IgG antibody (1:100 dilution; Molecular Probes, Eugene, OR, USA). Images were obtained using an LSM510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany) at the Central Research Laboratory at Okayama University Medical School in Okayama, Japan. For colocalization studies, both fluorophores were separated through careful selection of emission beam splitters and barrier filters. Signal bleed-through of eight probes was imaged using identical settings (iris, gain, and black level). Images from desmin (red) and α-SMA (green) fluorescence patterns were processed as one-color images or two-color overlays, as indicated.