Protease k

Renal biopsy was performed to investigate the cause of the nephrotic syndrome. Tissues for light microscopy and immunofluorescence were routinely fixed in formalin and embedded in paraffin. The formalin-fixed paraffin-embedded sections were cut at 2 μm and stained with hematoxylin and eosin, periodic acid-Schiff, periodic acid methenamine silver, Masson's trichrome, and Congo red. Immunofluorescence staining was performed using formalin-fixed paraffin-embedded sections that were prepared by protease K (Dako, Copenhagen, Denmark) for 60 min. Fluorescein isothiocyanate-labeled rabbit anti-human IgG, IgA, IgM, C1q, C3, fibrinogen, kappa- and lambda-light chains (Dako) were used. Additionally, mouse monoclonal anti-human IgG1, IgG2, IgG4 (Chemicon, Temecula, CA, USA), and IgG3 (Thermo Fisher Scientific) antibodies, and rabbit polyclonal anti-PLA2R antibodies (Abcam) were used followed by Alexa 546 goat anti-mouse IgG (H+L) and Alexa 488 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific).

EGFR was assessed by immunohistochemistry, IHC, using a streptavidin-biotin complex technique as previously described.23 (link),24 (link) Endogenous peroxidase was blocked in 0.3% H₂O₂ in PBS for 20 min. Then, antigen retrieval was done in 0.05% protease K (Code no. S3020, Dako, Glostrup, Denmark) in PBS for 10 min at room temperature. The tissue sections were preincubated in PBS for 10 min and then the primary mouse monoclonal antibody (clone 31G7, Zymed labs, South San Francisco, CA, USA) directed against the EGF receptor, diluted 1:100, was added and the sections incubated overnight at 4°C. The secondary biotinylated antibody (goat anti-mouse, Dako, Glostrup, Denmark) and the peroxidase-labeled streptavidin-biotin complex (Dako, Glostrup, Denmark), diluted 1:200, were then added and the samples incubated for 30 min at room temperature. All sections were developed in 0.05% diamino benzidine (Sigma, St Louis, MO, USA) for 5 min and counterstained in Harris haematoxylin (Sigma, St Louis, MO, USA). Finally, the sections were dehydrated through graded alcohol to xylene and mounted in organic mounting medium (Pertex®, Histolab, Gothenburg, Sweden).

Apoptotic cells were detected using an ApopTag Fluorescein In Situ Apoptosis Detection kit (Roche, Mannheim, Germany). Paraffin-embedded kidney slides were deparaffinized, hydrated, and sequentially incubated with protease K (Dako, Santa Clara, CA, USA), terminal deoxynucleotidyl transferase enzyme, and anti-digoxigenin-fluorescein (Roche). The slides were mounted with Gold antifade reagent containing 4',6-diamidino-2-phenylindole (Invitrogen, Carlsbad, Calif, USA) and examined under a fluorescence microscope. For each sample, five fields (400×) were randomly selected, and the staining was quantified using computer-based morphometric analysis (Qwin 3, Leica, Mannheim, Germany). Scoring was performed in a blinded manner using the mean values of the positive areas (%).