Anti-rabbit IgG conjugated with HRP was used as secondary antibody, and nickel enhanced diaminobenzidine
was used as HRP substrate.
Abbreviations DAB: Diaminobenzidine
H&E: Haematoxylin and Eosin IHC: Immunohistochemistry ER: Estrogen receptors HER2: Human epidermal growth factor receptor 2 OS: Overall survival PBS: Phosphate buffer saline PgR: Progesterone receptors PBS: Phosphate buffered saline RFS: Recurrence-free survival rTdT: Recombinant Terminal Deoxynucleotidyl Transferase TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling TNBC: Triple-negative breast cancer.
The Leica polymer and postpolymer were each incubated for 30 minutes, followed by 10 minutes in diaminobenzidine
, and a light hematoxylin counterstain.
Tissue preparation and diaminobenzidine
Specimens were then rinsed six to eight times over 10 h in PBS + Triton-X 100, followed by incubation for 30 min in diaminobenzidine
(solution provided in kit marketed by DAKO Diagnostics, Mississauga, ON, Canada) diluted into PBS.
The reaction was visualized by incubating the sections in a solution of 0.1 % 3,30 diaminobenzidine
(DAB) and 0.02 % [H.sub.2][O.sub.2] for 7 min (DAB substrate kit; Invitrogen).
Detection of the primary antibody or mouse IgG11j was performed using EnVision FLEX+ Polymer reagents (horseradish peroxidase polymer, diaminobenzidine
chromogen, and diaminobenzidine
enhancer), washing with EnVision FLEX+ wash buffer between the incubation steps.
Dulbecco's Modified Eagle Medium (DMEM), fetal calf serum (FCS), glutamine, [iota]-glutamic acid (monosodium salt), poly-D-lysine, diaminobenzidine
, propidium iodide (PI), pyruvic acid ([alpha]-ketopropionic acid) sodium salt, [beta]-nicotinamide adenine dinucleotide (reduced form), paraformaldehyde, 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl, free radical (DPPH) and ([+ or -])-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma (USA).
The secondary label was then revealed by the diaminobenzidine
(DAB) staining method (Hsu and Soban, 1982) using the Sigma Fast DAB peroxidase substrate tablet set (Sigma no.
Mouse monoclonal antibody clone 124 against bcl-2, rabbit anti-mouse IgG alkaline phosphatase conjugate, biotinylated rabbit anti-mouse antibody, the avidin-biotin-peroxidase complex kit, and diaminobenzidine
were purchased from Dako.
The reaction was visualized by incubating the sections for 7 min in a 0.1 % 3,3 diaminobenzidine
and 0.02 % hydrogen peroxide solution (DAB substrate kit, Invitrogen, Carlsbad CA).
Each strip was incubated with a 1:100 dilution of individual serum specimens in 0.3% Tween-PBS, peroxidase-conjugated goat antibody (Bio Source International Inc., Camarillo, CA, USA) diluted 1:4,000 in 0.05% Tween-PBS, and blots were developed by diaminobenzidine
substrate solution containing [H.sub.2][O.sub.2].