Reagents
Download the Apo-BrdU-IHC protocol booklet here
The Phoenix Flow Systems APO-BRDU-IHC reagent kit is a two color TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) assay for labeling DNA breaks to detect apoptotic cells by immunohistochemistry.The kit contains the instructions and reagents required for measuring apoptosis in cells including; positive/negative control slides for assessing reagent performance; reaction and blocking buffers for processing individual steps in the assay; proteinase K; terminal deoxynucleotidyl tranferase enzyme (TdT), bromodeoxyuridine triphosphate (Br-dUTP), biotin labeled anti-BrdU antibody for labeling DNA breaks, horseradish peroxidase streptavidin conjugate, DAB, H2O2/Urea tablets for color generation and methyl green solution for counterstaining the cells.
One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of “DNA laddering” when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells which remains largely intact does not display this “laddering” on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3’-hydroxyl ends in the DNA. This property can be used to identify apoptotic cells by labeling the 3’-hydroxyl ends of the DNA with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes this template independent addition of the Br-dUPT's to the 3’-hydroxyl ends of double- or single-stranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the APO-BRDU-IHC Kit. Non-apoptotic cells do not incorporate significant amounts of the Br-dUTP owing to the lack of exposed 3’-hydroxyl DNA ends. After labeling the 3’-hydroxyl ends of the DNA with Br-dUTP, an anti-BrdU antibody directly conjugated to biotin is attached to the Br-dUTP’s. Horseradish peroxidase conjugated to avidin is attached to the biotin labeled Br-dUTP’s. Then a standard contrast microscope stain reaction, DAB-H2O2-Urea, is used to label the incorporated Br-dUTP’s. Recent evidence has demonstrated that Br-dUTP is more readily incorporated into the genome of apoptotic cells than are the deoxynucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin (1). This greater incorporation gives rise to a stronger flow cytometry signal when the Br-dUTP sites are identified by a fluorescein labeled anti BrdU monoclonal antibody and may increase the signal to noise ratio in this immunohistochemical staining method.
Part No. AH-1001
50 Assay Kit plus 2 positive & 2 negative control slides
Kit Contents:
Positive & Negative Control Slides
Blocking Buffer
H202/Urea Tablets
Proteinase K
DAB Tablets
TdT Enzyme
Br-dUTP
200X Conjugate
5X-Reaction Buffer
Biotin-anti BrdU antibody
Methyl Green