Intended use: The Plus AP Polymer anti-Mouse is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with monoclonal primary antibodies obtained from mouse. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
Summary and explanation: The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer anti-Mouse is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The reagent is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mouse. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer anti-Mouse avoids the problem of background staining caused by endogenous biotin in the tissue.
Reagents provided: 6 ml AP-Polymer anti-Mouse (Ready-to-use) Substrate systems recommended: Permanent AP Red kit, Fast Red substrate kit, New Fuchsin kit.Materials required but not supplied:Positive und negative control tissueXylene or suitable substitutesEthanol, distilled H2OReagents for enzyme digestion or heat pre-treatmentWash buffer PBS or TBS PAP Pen Primary antibody (user-defined)Primary antibody diluent Negative control reagentChromogenic substrateCounter stain solutionMounting mediumCover slips
Storage and handling: The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Principle of method: Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (“Blocking Solution”). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP-polymer is applied and incubated. Any excess of unbound AP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Fast Red leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red), New Fuchsin (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagent preparation: Reagents should be at room temperature when used. • Deparaffinise and rehydrate paraffin-embedded tissue sections. • Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. • Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure: 1. Blocking Solution (protein block, this step is optional) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP-polymer anti Mouse 30 min. 6. Washing with wash buffer 3 x 2 min. 7. Fast Red, Permanent AP Red, NBT/BCIP or New Fuchsin 5-15 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: aqueous with Fast Red, permanent with Permanent AP Red, NBT/BCIP or New Fuchsin
Expected results: During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
References: Elias JM Immunohistopathology – A practical Approach to Diagnosis ASCP Press 2003/Nadji M and Morales AR Ann N.Y. Acad Sci 420:134-139, 1983
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