I am performing IHC on tissue sections using a mouse IgG primary antibody. My detection system is a biotinylated anti-mouse IgG secondary antibody, a VECTASTAIN® Elite ABC peroxidase kit (PK-6100), in combination with a DAB substrate kit (SK-4100), and I do not see any staining. How do I know if the end detection reagents are working?
Make a fresh working solution of DAB substrate per instructions. Place a small volume (~1 mL) of this DAB substrate into a clean glass test tube. To this 1 ml aliquot, add one drop (~50 µL) of Reagent B only from the VECTASTAIN® Elite ABC kit. If the HRP enzyme is active and the DAB reacts with it, an immediate color change will be observed. This indicates the end detection reagents are working appropriately.