Intended use: The Plus AP Polymer Kit 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 mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. The kit 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 Kit is a highly sensitive detection kit 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 test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer Kit avoids the problem of background staining caused by endogenous biotin in the tissue.
Reagents provided: 100 ml Blocking Solution Reagent 1 (ready-to-use) 100 ml PostBlock Reagent 2 (ready-to-use)100 ml AP-Polymer (Mouse/Rabbit) Reagent 3 (ready-to-use) Materials required but not suppliedPositive 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 solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They 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”, provided with this kit). 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 enhancement reagent (“PostBlock“) is applied and incubated. A second washing is followed by the application of the AP-polymer. Any excess of unbound APpolymer 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 Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
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, Reagent 1) (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. PostBlock (Reagent 2, yellow) 20 min. 6. Washing with wash buffer 3 x 5 min. 7. AP-polymer (Reagent 3, red) 30 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent AP Red 10-20 min. (Controlling the colour intensity via light microscope is recommended.) 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: permanent or aqueous with Permanent AP Red
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/Omata M et al. Am J Clin Pathol 73: 626-632, 1980
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