Table 1 Details on immunohistochemical procedures
Cell type | Tissue processing | Marker | Antibody | Conjugate(s) + visualization |
|---|---|---|---|---|
Macrophages + collagen III | Cryosections, acetone fixed | Collagen III + F4/80 | Rat anti-mouse F4/80 (eBioscience, CA, USA) 1:1500 + goat anti-collagen III (Southern Biotech, Birmingham, USA), 1:40 | Donkey anti-Rat IgG Alexa 594 (Molecular Probes, Eugene, OR, USA) 1:250 + donkey anti-goat IgG FITC (Southern Biotech, Birmingham, USA); 1:50. Tetramethylrhodamine System (PerkinElmer LAS Inc.) |
T-cells | Formaldehyde fixed, paraffin embedded, deparaffinization, Tris/EDTA (pH 9.0) | CD3 | Rabbit anti-human CD-3 (cross-reactive with mouse; DAKO, Glostrup, Denmark) 1:100 | Goat anti-rabbit Ig HRP 1:100. rabbit anti-goat Ig HRP (DAKO, Glostrup, Denmark) 1:100. Tetramethylrhodamine System (PerkinElmer LAS Inc) |
Neutrophils | Cryosections, acetone fixed | NimpR14 | Rat anti-mouse NimpR14 (Abcam, Cambridge, UK) 1:2000 | Rabbit anti-rat Ig HRP (DAKO, Glostrup, Denmark) 1:100. 3-amino-9-ethyl-carbazole (AEC) |
Myofibroblasts | Cryosections, acetone fixed | αSMA | Mouse anti Rabbit anti-SMA 1A4 (Sigma, Zwijndrecht, The Netherlands) 1:4000 | Goat α mouse IgG2a HRP (Southern Biotech, Birmingham, USA) 1:100+ rabbit anti-goat Ig HRP (DAKO, Glostrup, Denmark) 1:100. Tetramethylrhodamine System (PerkinElmer LAS Inc) |
Collagen III | Cryosections, acetone fixed | Collagen III | Goat anti-collagen III (Southern Biotech, Birmingham, USA) 1:50 | Rabbit anti-goat Ig HRP 1:100. Goat anti-rabbit Ig HRP (DAKO, Glostrup, Denmark) 1:100. 3-amino-9-ethyl-carbazole (AEC) |
Podocytes | Cryosections, acetone fixed | Wilms’ tumor protein 1 | Rabbit anti-human WT-1 C-19 (Santa Cruz Biotechnology) 1:100 | Goat anti-rabbit Ig HRP 1:100 + rabbit anti-goat Ig HRP 1:100 (DAKO, Glostrup, Denmark). 3-amino-9-ethyl-carbazole (AEC) |
MBL-C + Agrin | Cryosections, acetone fixed | MBL-C | Rat anti-mouse MBL-C (Hycult biotech, Uden, the Netherlands) 1:100 + sheep anti-agrin (GR14, own stock), 1:1500 | Goat anti-rat IgG HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) 1:500 + rabbit anti-sheep IgG FITC (Abcam, Cambridge, UK); 1:50. Tetramethylrhodamine System (PerkinElmer LAS Inc) |
Complement activation | Cryosections, acetone fixed | C3b/iC3b/C3c | Rat anti-mouse C3b (Hycult biotech, Uden, the Netherlands) 1:100 | Rabbit anti-rat Ig HRP 1:100 + goat anti-rabbit Ig HRP (DAKO, Glostrup, Denmark) 1:100. 3-amino-9-ethyl-carbazole (AEC) |
