Pathology samples are routinely fixed in formaldehyde, embedded in paraffin blocks (FFPE), cut into sections, stained with dyes and viewed under a microscope. This method preserves cellular structure and reveals considerable detail. Such blocks are collected over many years and thus provide an invaluable clinical database for studies on gene expression. This method is of particular utility in studies of protein expression in human tumor surgical samples. There are now many examples where the detection of particular proteins directs a specific therapy. For example the overexpression of HER-2 prompts treatment with Trastuzumab
However, this fixation method cross-links proteins and thus these sections do not work well in standard immunohistochemical assays. In most cases the epitope recognized by a particular the cross-linked residues obscure antibody and little reactivity is possible. To reactivate the protein antigen, the sections are heated in a buffer, which removes the crosslinks. Some protocols recommend steam heat some recommend microwave. Clonegene has now launched a new retrieval reagent called RETREVIA. The above picture shows the strong reactivity of a lung cancer section with our anti-HuR antibody (CG001) after treatment with RETREVIA .
Below we summarize the key steps that we recommend to give superior staining with our antibodies
1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.
2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water.
3. Pre-heat steamer or water bath with staining dish containing RETREVIA (dilute 10 ml of RETREVIA to 1 Litre of distilled water) until temperature reaches 95-100 °C.
4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 30 minutes (however this incubation time may depend on the nature of the sections and should be determined by user).
5. Turn off steamer or water bath and remove the staining dishes to room temperature and allow the slides to cool for 20 minutes.
6. Rinse sections in PBS for 2x2 min.
Proceed to standard immunohistochemistry protocol, we recommend Vectors labs ABC elite staining kit.
RETREVIA also works well in automated staining platforms.
For sales or other information contact Clonegene