For IHC, antibody binding was detected by using the "Envision+" System-HRP (DAB) followed by chromogen detection with diaminobenzidine (DAKO).
In brief, digoxigenin-dUTP end labeled DNA was detected with anti-digoxigenin-peroxidase antibody followed by peroxidase detection with diaminobenzidine (DAB).
The AQP5 antibody was visualized using the avidin-biotin-peroxidase technique (DAKO LSAB kit; DAKO Cytomation, Carpinteria, CA) and followed by chromogen detection with diaminobenzidine (DAB).
After intermittent rinses in TBS, avidin biotin peroxidase complex was applied (ABC Elite kit, Vector Labs, Burlingame, CA, USA) followed by peroxidase detection with diaminobenzidine in 0.01% H2O2, 0.04% NiCl in TBS.
This was followed by incubation with appropriate biotinylated secondary antibody, followed by reaction with streptavidin-peroxidase, detection with diaminobenzidine (DAB, Sigma D5905), and finally counterstaining with hematoxylin.
Primary antibodies underwent 1 h incubation at room temperature followed by incubation with appropriate biotinylated anti-rabbit IgG antibody, followed by reaction with streptavidin-peroxidase, detection with diaminobenzidine (DAB, Sigma D5905, St . Louis USA), and finally counterstaining with hematoxylin.
antibodies, followed by incubation with HRP (DAKO, Glostrup, Denmark) by using the EnVision+ System, and detection with diaminobenzidine.
Specimens were incubated with terminal deoxynucleotidyl transferase/bromide- deoxynucleoside triphosphate mixture, followed by anti-BrdU treatment and incubation in streptavidin-horseradish peroxidase, then subsequent detection with diaminobenzidine.
with horseradish peroxidase-labeled streptavidin (Zymed, Inc ., and detection with diaminobenzidine.
Immunostaining was performed with the Lab Vision Autostainer (Lab Vision, Fremont, USA) using a biotin-streptavidin-peroxidase detection system with diaminobenzidine as a chromogen (ChemMate™ Detection kit, DakoCytomation).
