Envision kit

EDTA-decalcified, paraffin-embedded tissue sections were immunostained with affinity purified rabbit anti-human ClC-7 (Abcam, catalogue ab31264) and rabbit anti-Ae2 (courtesy Dr. S. Kellokumpu, University of Oulu, Finland). According to the manufacturer, anti-ClC-7 antibody was raised against a synthetic peptide-conjugate to KLH from within amino acid residues 750 to C-terminus of the human ClC-7, with a predicted 94 % identity with mouse, rat and rabbit. Sections were deparaffinized in xylene, rehydrated in ethanol and washed in Tris-buffered saline (TBS). Antigen retrieval was carried out for both antibodies by incubation in 10 mM citrate buffer (pH 6.0) overnight at 60 °C. Nonspecific staining of the tissues was blocked by 30 min incubation with normal goat sera. Sections were then incubated with primary antibodies (1:200 dilution for ClC-7 antibody, 1:100 dilution for the anti-Ae2 antibody) or non-immune rabbit IgG (negative controls) overnight at 4 °C. After washing in TBS, sections were incubated with goat anti-rabbit-IgG-polymer conjugated with peroxidase (EnVision kit; Dako Cytomation, Glostrup, Denmark) for 1 h at ambient temperature (Henriksen et al. 2004 (link); Schaller et al. 2004 (link)), washed and the peroxidase visualized using DAB (EnVision kit), counterstained with hematoxylin, dehydrated and mounted in Depex.

Consecutive sections (4-μm) of tissue microarray samples were mounted on the APES-coated slides. After de-paraffinization, rehydration and antigen retrieval using an autoclave-oven-technique, sections were incubated with anti-PRR11 (HPA023923, SIGMA-ALDRICH), UCHL1 (HPA005993, SIGMA-ALDRICH), SNAT1 (ab59721, abcam®), and EGR1 (ab54966, abcam®) at 4°C overnight. A two-step EnVisionTM KIT (DAKO, USA) was used to visualize positive staining. Lung cancers known to be positive for these proteins were used as positive controls. Replacement of the primary antibody by PBS served as a negative control.

Sections (5 μm) of the specimens were incubated with mouse anti-human primary antibodies (i.e. anti-BRG1, α-SMA, MMP2 and MMP9, Santa Cruz) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse antibody (Santa Cruz) for 1 hr at 37°C. Immunodetection was performed with the EnVision^TM Kit (Dako), using diaminobenzidine as the chromogen.

The cartilage explants (125 mm³) were fixed in 4% paraformaldehyde in PBS for 24 h at 4°C, washed with PBS, dehydrated with graded ethanol, embedded in paraffin and sectioned into 4-μm slices as described previously (28 (link)). The sections were stained by the standard procedures using antibodies against type II collagen or COX-2, and visualized by development with an EnVisionTM+ kit purchased from Dako (Carpinteria, CA, USA) following the manufacturer’s instructions.

Pathological changes were studied by light microscopy on four-micrometer thick sections of formalin-fixed, paraffin-embedded tissue samples stained with haematoxylin and eosin. Immunohistochemical examinations were performed as described before [22 (link)]. In brief, sections were de-paraffinized and incubated in 3% H₂O₂ solution for 10 min and in 2% solution of skimmed milk powder for 20 min. The sections were incubated overnight at 4 °C with anti-F. tularensis hyperimmune rabbit serum diluted 1:30,000 in phosphate-buffered saline (PBS). Antibody binding was detected using anti-rabbit antibodies with horse-radish peroxidase (HRP)-labelled polymer following manufacturer’s instructions (EnVisionTM + Kit; Dako, Denmark). Non-infected rat tissue samples served as a negative control. For some sections antiserum was replaced by PBS to rule out the possibility of any nonspecific binding. Lesions and immunoreactivity were evaluated by a single pathologist in a treatment-blinded manner. The amount of antigen in the lung, liver and spleen was graded based on the number of affected organs and antigen observations/field of vision at 100x magnification (scored as minimal: <10 observations in only one organ; low: <10 observations in two or three organs; moderate: 10–20 observations in two or three organs; high: >20 observations in all three organs).

The paraffin blocks were sectioned at 5μm and stained with haematoxylin and eosin. Anti-HLA A antibody (Abcam) and Envision^TM+ Kit (Dako, Glostrup, Denmark) were used for detecting HLA-A.