Depicted here is a simple schematic showing the process of IHC. First, a tissue is incubated with a primary antibody, known to bind a specific antigen. Then, a secondary antibody is used to bind to the primary antibody in order to enhance the final signal. Third, an additional antibody is added which binds to the secondary antibody and contains a visualization probe, such as horseradish peroxidase. Finally, the visualization probe is activated in order to see exactly the amount of, and where a specific antigen is located.
Technical manual for IHC method
Phase I: The process of generating a MonoRab™ custom rabbit mAb begins by immunizing rabbits with the desired immunogen, which can be a peptide, protein, DNA, Cell line or corresponding to the intended antigen. GenScript can also recombinantly generate the antigen if the client does not have it already purified.
Phase II: The immunized rabbits will go through multiple rounds of additional immunogen boosts, test bleeds, and ELISA screenings until the serum is positive for a high titer. Serum purified and the pAb tested for IHC application at GenScript on rodent tissue, human tissue or client desired tissue type. Based on pAb IHC staining and signal intensity, the fusion can be performed, otherwise we give additional boosts to get satisfactory pAb IHC data.
Phase III: After immunization and screening, individual B-cells are isolated from 3 rabbits and electro-fused to mouse myeloma cells to generate hybridoma cells. Three hybridomas are then subcloned and screened for positive monoclones. The supernatants of these monoclones are used in ELISA screenings as well as customer analysis.
Phase IV: After the selection of a specific clone for mAb generation, the B-cell is subjected to antibody variable region sequencing.
Phase V: Variable region sequencing data is used for recombinant expression and purification of the desired mAb.