Immunohistochemical examination on mouse tissue is often difficult because a lot of primary antibodies are mouse-based and lead to high background when applied on mouse tissue. Millipore offers a quick and efficient solution for this problem: The mouse-to-mouse detection system is designed for visualizing mouse antibodies on mouse or rat tissue. It also works with rat or rabbit primary antibodies. The main features are: It is a biotin-free system, is ready-to-use (except the DAB solution), has a high sensitivity and low background. It utilizes a new controlled polymerization technology to prepare HRP-linked antibody conjugates. You can choose either DAB or AEC as substrate.
I often use the kit for my immunohistochemistry examinations, at least twice a month. Application works on paraformaldehye fixed (24 h, 4% in PBS), paraffin embedded tissue or shock-frozen tissue in OCT/tissue tek. I do not do large scale stainings; that’s why I only order the 7 ml quantity. But the kit is also available with 15 ml or as a small trial set with 3 ml.
The manufacturer’s protocol is very useful and I only varied the dilution medium for primary antibody; otherwise I followed the recommended procedure. First, I deparaffinize my slides followed by rehydration. Then I perform antigen retrieval in the microwave. Afterwards, I block endogenous peroxidase with 3% H2O2 in ddH20 for 10 minutes. The slides are washed for 5 min in PBS and the PRE-antibody blocking solution is applied for 20 min. The solution is blotted off with filter tissue because there is no need to wash. Then the slides are incubated with mouse primary antibody overnight at 4°C. Alternatively, the slides can be incubated at room temperature for 30-60 minutes. I dilute the antibody in antibody diluent (serum free) from DAKO. Next, the slides are washed with PBS-Tween (0,05 %) twice for 5 minutes. They are again blocked with POST-antibody blocking solution for 10 minutes and washed with PBS-Tween twice for 5 minutes. Then the ready-to-use Poly-HRP-Anti-Mouse/Rabbit IgG solution is applied for 10 minutes, followed by washing with PBS-Tween for 5 minutes, two times.
Finally, I prepare the provided DAB solution with 940 µl ddH20. It is important to mix this solution immediately prior to use and it cannot be used again. DAB is applied until staining is detected and the slides are washed with distilled water for 1-2 minutes. Counterstaining can be performed with haematoxylin. All procedures except the primary antibody incubation are performed at room temperature. The antibodies I applied were against different epitopes, e.g. in the nucleus, on the surface or in cytosol of cells and the system worked always fine. Some of the treated mouse tissue included uterus, ovary, gut and peritoneum.
The only disadvantage is the sensitive enzyme (HRP) solution – you have to pay attention to the expiry date. Moreover, the PRE-antibody blocking solution seems to run out faster than the other components of the kit, so do not waste it and apply it carefully.
In sum, I can highly recommend this very convenient and fast visualization system.