What are the advantages of using ImmPRESS^® Excel kits compared with one-step peroxidase polymer detection systems?

The ImmPRESS Excel kits are a two-step peroxidase polymer based detection system that include an unconjugated amplifier antibody. This intermediate amplifier antibody increases sensitivity of the assay at least three- to four-fold over that of a one-step polymer based system, by facilitating the introduction of more peroxidase enzyme at the site of specific antigen localization. This increase in sensitivity would be advantageous in instances of weak antigen expression, and to further dilute out an expensive primary antibody. The ImmPRESS Excel Amplifier kits are presented as a complete kit format that include enzyme quench, blocking serum and ImmPACT^® DAB EqV peroxidase substrate, in addition to the amplifier antibody and defined ImmPRESS polymer secondary antibody. However, as the two published references below indicate, this format is still modular and allows for the substitution of different detection reagents. These references describe using ImmPACT NovaRED™ peroxidase substrate in combination with the ImmPRESS Excel Amplifier kits. Oncogenesis (2017) 6, e293; doi:10.1038/oncsis.2016.82 Tan, E.M.S., et al (2017) Front. Med. 4:162 (doi: 10.3389/fmed.2017.00162)

Our research lab has an open automated staining platform. Do you know if the ImmPRESS reagents can be applied to an automated system, and if so, do you have any guidelines or procedures?

Yes, we conducted studies on the compatibility of the ImmPRESS HRP polymer reagents with three commercially available autostainers (Agilent/Dako Autostainer Plus, Leica Bond Rx, and Ventana Discovery Ultra). We showed that our reagents are suitable for IHC detection on each of these platforms. The ImmPRESS polymer reagents and enzyme substrates generated equivalent IHC staining results compared to reagents from the instrument manufacturer. Some modifications in the protocol were performed to optimize the signal-to-noise ratio, such as a shorter incubation time for the ImmPRESS polymer and increase in the number of buffer washes following polymer incubation. Please see the following application note for more details about each automated platform:https://info.vectorlabs.com/l/360741/2019-01-11/2hv9mw

The staining procedure is simple as shown in the diagram below. Following a blocking step with the diluted normal horse serum, sections are incubated with primary antibody. After a brief wash, the appropriate ImmPRESS Reagent is added to the sections and incubated for 30 minutes. Sections are again rinsed and the slides are developed with the peroxidase substrate of choice.

When choosing the optimal detection system for your application, it is important to consider not only the species of the primary antibody but also the species of the tissue. If the species of the primary antibody and the species of the tissue are closely related (e.g. rat and mouse), the secondary antibody may bind to endogenous IgG in the tissue section leading to background. The following options minimize background staining in these instances:

• Use a secondary antibody specifically adsorbed to remove cross-reacting antibodies of closely-related species (e.g. ImmPRESS Anti-Mouse IgG, Rat Adsorbed).
• Use the M.O.M.^® ImmPRESS Kit (Cat. No. MP-2400) for applications of mouse primary antibodies on mouse tissue.