Immunohistochemistry of mouse trachea and lungs.

Mouse trachea and lungs were stained for acid ceramidase, sphingosine, or ceramide as previously described (11,–13). Mice were sacrificed by cervical dislocation, and the trachea and lungs were removed and immediately fixed in 4% phosphate-buffered saline (PBS)-buffered paraformaldehyde (PFA) (catalog number 0335.3; Roth). The trachea was opened prior to fixation. Fixation was performed for 48 h. The tissue was serially dehydrated with an ethanol-to-xylol gradient, embedded in paraffin, sectioned at 7 μm, dewaxed, and rehydrated. To retrieve the antigens, sections were treated with pepsin (Digest All, catalog number 003009; Invitrogen) for 30 min at 37°C. The sections were then washed, blocked for 10 min at room temperature with PBS, supplemented with 5% fetal calf serum (FCS), washed once in PBS, and stained with anti-acid ceramidase (1:100 dilution) (catalog number 4741; ProSci, Heidelberg, Germany), anti-ceramide (1:200 dilution) (clone S58-9, catalog number MAB_0011; Glycobiotech), anti-sphingosine (1:1,000 dilution) (clone NHSPH, catalog number ALF-274042010; Alfresa Pharma Corporation, Osaka, Japan), or anti-mouse β1-integrin (clone MB1.2, catalog number MAB1997; Merck Millipore) antibody at room temperature for 45 min. Antibodies were diluted in HEPES/Saline (H/S) consisting of 132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl₂, 0.7 mM MgCl₂, 0.8 mM MgSO₄ plus 1% FCS. The sections were washed three times for 5 min each with PBS plus 0.05% Tween 20 and once with PBS. The tissues were then secondarily labeled with Cy3-coupled anti-rabbit IgG (for anti-acid ceramidase antibodies), Cy3-coupled anti-mouse IgM (for anti-sphingosine or anti-ceramide antibodies), or anti-rat IgG (for anti-β1-integrin antibodies) F(ab)₂ fragments (Jackson Immunoresearch) in H/S plus 1% FCS for 30 min. Sections were washed three times as described above and once with PBS and embedded in Mowiol. Samples were evaluated by confocal microscopy on a Leica TCS-SL confocal microscope equipped with a 40× lens, and images were analyzed with Leica LCS software (Leica Microsystems, Mannheim, Germany). All comparative samples were measured at identical settings. Control stainings were performed with Cy3-labeled secondary antibodies only or with irrelevant rabbit IgG, mouse IgM, or rat IgG, followed by staining with the corresponding Cy3-labeled secondary antibodies. These controls revealed very weak staining and confirmed the specificity of the antibody staining. Fluorescence intensities were quantified using ImageJ.