Intended use: The Fast AP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.It is intended for in vitro diagnostic use.
Summary and explanation: The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Fast AP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzymesubstrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. Cross-reactivity with primary antibodies from rat has been observed. In contrast to other detection techniques, which often use the streptavidin-biotin system the Fast AP One-Step Polymer anti-Mouse/Rabbit avoids the problem of background staining caused by endogenous biotin in the tissue.
Reagents provided: 6 ml Fast AP One-Step Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanaent AP Red kit Materials required but not suppliedPositive and negative control tissueXylene or suitable substitutesEthanol, distilled H2OReagents for enzyme digestion or heat pre-treatmentWash buffer PBS or TBSBlocking Solution (for protein blocking, optional)Pink PAP Pen Primary antibody (user-defined)Primary antibody diluentNegative control reagentChromogenic substrateCounter stain solutionMounting mediumCover slips
Storage and handling: The solution should be stored at 2-8°C without further 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. 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 reagent, 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 OneStep Polymer is minimized by incubation with a protein 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 One-Step Polymer is applied and incubated. Any excess of unbound 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 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 (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 One-Step Polymer anti-Mouse/Rabbit 30 Min. 6. Washing with wash buffer 3 x 2 min. 7. Permanent AP Red, 10-20 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: permanent or aqueous with Permanent AP Red Kit
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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