Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections, as described previously.
22 23 Briefly, deparaffinized sections were soaked in 0.3% hydrogen peroxidase in methanol for 30 minutes at room temperature to inactivate endogenous peroxidase activity. The sections were then blocked with 10% goat serum for 1 hour and then incubated overnight at 4°C with the following primary antibodies: mouse anti-human HSP47 (StressGen Biotechnologies Corp., Victoria, BC, Canada), mouse anti-human type I collagen, mouse anti-human type III collagen (Daiichi Fine Chemicals, Takaoka City, Japan), or mouse anti-human α-SMA (Dako, Glostrup, Denmark). Normal mouse serum was used as a negative control. After being washed with PBS, the sections were treated with a biotinylated mouse secondary antibody for 15 minutes, washed with PBS, and incubated further with streptavidin-peroxidase. The reaction products were developed with a mixture of 3,3′-diaminobenzine-4 HCl (DAB) and H
2O
2. The blocking solution, secondary antibody, and streptavidin-peroxidase solution were from a histology kit (Histostain kit; Nichirei, Tokyo, Japan). The staining pattern was graded semiquantitatively according to the intensity and distribution of the staining, as described in our earlier reports.
22 23 Positive staining for HSP47, collagens and α-SMA was determined by comparison with smooth muscle cells of the vessels or myoepithelium in the same section as a positive reference. We regarded the expression as positive when more than one cell showed a staining intensity similar to that of the reference cells. The staining intensity of HSP47, collagen types I and III, and α-SMA was graded semiquantitatively according to the following scale: (−) no staining, (+) moderate staining, and (++) strong staining.
To double-stain for HSP47 and Ki67 in paraffin-embedded tissue sections, we performed antigen unmasking by autoclaving the sections at 120°C for 20 minutes in 10 mM sodium citrate buffer (pH 6.0). For the immune reaction, we used a mouse anti-HSP47 antibody (Stressgen Biotechnologies Corp.) followed by an Alexa 568-conjugated rabbit anti-mouse secondary antibody (Invitrogen-Molecular Probes, Eugene, OR) and a FITC-conjugated mouse anti-Ki67 antibody (Dako).
Fibroblasts were cultured on fibronectin-coated chamber slides, on which they were fixed with 10% neutral buffered formalin for DAB staining or with 4% paraformaldehyde for fluorescent staining. For DAB staining of cultured fibroblasts, the endogenous peroxidase activity was quenched with 0.3% peroxide for 5 minutes. A mouse anti-human HSP47 (Stress Gen Biotechnologies Corp.), mouse anti-human type I collagen, or mouse anti-human α-SMA antibodies (mAb) and peroxidase-conjugated secondary Ab (En Vision+; Dako, Glostrup, Denmark) were used in combination with nuclear staining with hematoxylin. The negative controls were cells incubated with normal mouse IgG instead of the primary antibody. At least 300 cells per culture were evaluate, to enumerate the proportion of cells staining positive for each protein.
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For fluorescent staining, the coexpression in cultured fibroblasts of HSP47, collagen types I and III, and α-SMA was examined by double-staining with a rabbit anti-HSP47 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) used with an Alexa 488-conjugated goat anti-rabbit secondary antibody (Invitrogen-Molecular Probes), or with a mouse anti-collagen type I, anti-collagen type III, or anti-α-SMA (Dako) antibody used with an Alexa 568-conjugated rabbit anti-mouse secondary antibody (Invitrogen-Molecular Probes). Nuclei were counterstained with TO-PRO-3 (Invitrogen-Molecular Probes). Isotype-matched mouse antibodies were used as control subjects. These tissue sections and the cultured fibroblasts used for fluorescent staining were mounted on glass slides and examined with a confocal microscope (LSM5 Pascal; Carl Zeiss Meditec, GmbH, Göttingen, Germany).