Analysis of eNOS, iNOS, IL-1ß and ET-1 protein expression on human donor kidney biopsies
Immunohistochemical studies were performed on 2 µm sections of formalin-fixed, paraffin embedded human donor kidney biopsies, which were obtained immediately before transplantation.
- Slides were deparaffinized and rehydrated: 10 minutes in xylol, 10 minutes in 100% ethanol; 96, 70, 50% ethanol and aqua dest. for a few seconds each
- To inactivate endogenous peroxidase, tissue sections were incubated with 3% H2O2 for 10 minutes and rinsed with phosphate buffered saline (1xPBS, GibcoBRL) thereafter
- Antigen retrieval was carried out by autoclaving for 20 minutes at 1 bar in citrate buffer (citrat-monohydrate 0.01 M, pH 6) (Merck, Darmstadt, Germany), followed by cooling with aqua dest.
- Sections were blocked with avidin and biotin (Avidin-Biotin Blocking Kit; Vector laboratories, Burlingame, CA, USA) for 10 minutes each and rinsed with PBS in between
- Specimens were incubated with the following primary antibodies for 30 minutes; all antibodies were diluted in solutions of PBS containing 1% BSA:
eNOS/NOS Type III 1:2000, isotyp: IgG1 (mouse) (BD Biosciences, CA, USA)
NOS2 1:25, isotyp: IgG1 (mouse) (Santa Cruz Biotechnology, CA, USA)
ET-1 1:50, isotyp: IgG1 (mouse) (Affinity Bioreagents, CO, USA)
IL-1ß 1:200, isotyp: whole IgG (rabbit) (Abcam, Cambridge, UK)
- Slides were rinsed four times with PBS and the incubated with the second antibody (Super Stain Anti-Polyvalent, Super Stain System, ID Labs, USA) for 10 minutes
- After rinsing four times with PBS, sections were incubated with labeling reagent (Super Stain HRP, Super Stain System, ID Labs, USA) for 10 minutes
- Three washing steps with PBS were then followed by staining with AEC (ID Labs, USA)
- Staining was followed under the microscope and reaction stopped with distilled water
- Counterstaining was performed with hematoxylin for 4 minutes
- For microscopic investigations slides were mounted with aqueous mounting media (Aquatex, Merck, Darmstadt, Germany) and covered with glass slips
Sections of acute rejecting human kidney biopsies served as positive controls. Negative control experiments were performed in the absence of the primary antibody.