Description
BD Retrievagen A (pH 6.0) is an antigen retrieval system that is recommended for Zinc-fixed and Formalin-fixed paraffin embedded tissue sections that show sub-optimal or no reactivity with antibodies for immunohistochemical staining. 10% neutral buffered formalin fixes the tissue by cross-linking proteins that may cause a conformational change or "masking" of the antigenic epitope leading to reduced or absent antibody reactivity. The BD Retrievagen A (pH 6.0) is designed to unmask the epitope and cause the enhancement of antibody staining. The proposed mechanism by which most of the antigen retrieval systems work is through breaking the cross-linking bonds formed between the protein molecules.
It is important to realize that in spite of antigen retrieval, some antibodies do not stain on formalin-fixed paraffin embedded sections. In these situations fixing the tissue in a less harsh fixative like the IHC zinc fixative developed by BD Pharmingen (Cat. No. 550523) may be helpful. In many cases our zinc-fixative preserves the epitope leading to good antibody staining at the same time depicting good tissue morphology. However, there are a few antigens that are not preserved by any gentle fixation, and in these situations use of fresh frozen tissue for immunohistochemical staining is the only alternative.
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Preparation and Storage
Store undiluted at 4°C.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
BD Pharmingen Retrievagen A (pH 6.0) is provided as two separate solutions that need to be mixed. Take 18 ml of BD Retrievagen A solution 1 and 82 ml of BD Retrievagen solution 2 and bring up the volume to 1 litre with distilled water. The pH of the final working solution should be around 6.0. This working solution can be stored at 4° C for up to a month. Antigen retrieval protocol is performed after deparaffinizing the slides as outlined below.
1.Deparaffinize slides (after drying thoroughly overnight at RT) in 2 changes of xylene for 10 min each.
2.Wash slides in 100% alcohol twice for 2 min each.
3.Block endogenous peroxidase activity by incubating for 10 min in 3% H2O2. Rinse 2-3 times in water.
4.Place slides in a plastic coplin jar or staining dish with the BD Retrievagen A working solution and heat to 193°F in a microwave.
5.Mix the solution and incubate the slides at the desired temperature of 193°F for 10 min.
6.Remove the staining dish, cover and allow the slides to slowly cool to room temperature for 20 min.
7.Wash slides with 2-3 changes of water and continue with the blocking with normal serum and application of the primary antibody.
