HistoReveal (15ml) (ab103720)

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HistoReveal (15ml) (ab103720)

    Overview

    • Product name

      HistoReveal (15ml)

    • General notes

      Proteolytic antigen retrieval (PIER) reagent HistoReveal (ab103720). Superior staining to conventional enzymes (eg. trypsin). Only 5 min incubation. Single stable solution, designed to unmask immunoreactive sites altered by fixation and/or the embedding process.

      The most commonly used tissue fixatives are aldehydes, such as formalin, which work by protein crosss-linking. Proper fixation of a specimen prevents antigen from being washed out and is a critical aspect of successful IHC staining. Cross-linking can mask antigens, preventing primary antibodies from binding, thus yielding weak staining or false negative results. Immunoglobulins are especially susceptible to formalin fixation.

       

      Treatment with a proteolytic enzyme is required to digest excess aldehyde linkages and expose the antigen. HistoReveal is a proteolytic enzyme solution that can be used in place of traditionally used enzyme pretreatments, such as pepsin or trypsin, and results in superior staining. Unlike other enzymes, HistoReveal is gentle on tissue and does not destroy morphology.

       

      Moreover HistoReveal requires only a brief incubation at room temperature. Performing antigen recovery with HistoReveal will allow you to dilute the primary antibody 5 to 10 fold further and achieve optimal staining.

       

      Digestion Procedure

      • Deparaffinize tissue sections and bring them into buffer.
      • Remove excess buffer from slides without letting tissue dry.
      • Depending on tissue section, add 1 or 2 drops of HistoReveal.
      • Incubate slides for 5 minutes at room temperature.
      • Wash slides with buffer and proceed with immunostaining.

    Properties

    Associated products

      Images

      • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HistoReveal (15ml) (ab103720)
        Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HistoReveal (15ml) (ab103720)
        Cytokeratin 20 being stained in colon tissue following antigen retrieval using HistoReveal.

      Protocols

      To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

      Click here to view the general protocols

      References (2)

      Publishing research using ab103720? Please let us know so that we can cite the reference in this datasheet.

      ab103720 has been referenced in 2 publications.

      • Linkous A  et al. Modeling Patient-Derived Glioblastoma with Cerebral Organoids. Cell Rep 26:3203-3211.e5 (2019). PubMed: 30893594
      • Sung CK  et al. A mouse polyomavirus-encoded microRNA targets the cellular apoptosis pathway through Smad2 inhibition. Virology 468-470C:57-62 (2014). PubMed: 25146733

      Customer reviews and Q&As

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      Question

      What is the protocol for this product?

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      Answer

      Thank you for contacting us.


      I have placed the protocol below. This product is to be used for antigen retrieval in place of pepsin or trypsin for enzymatic retrieval.

      PROCEDURE:

      1. Deparaffinize tissue sections and bring them to buffer

      2. Remove Excess buffer from slides without letting tissue sections get dry

      3. Depending on tissue section size add 1 to 2 drops of Historeveal

      4. Incubate slides for 5 min at RT

      5. Wash slides with buffer and proceed with immunostaining


      I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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      Question

      Hi! Thank you for your reply! I will try to detect myofibroblasts in cancer FFPE tissue using an antibody against alpha-SMA (ab7817) and a Alexa fluor-secondary antibody. To try to investigate the cell-type specific expression in this case I will use laser capture microdissection, extract RNA from the collected cells and run a qPCR on a small number of genes. I know that the RNA is likely to be degraded due to the fixation method but I will try and hope for the best. At least the FFPE tissue we have is fairly new and have been stored in 4°C. Most people in my lab use heat-induced antigen retrieval techiques for IF and IHC but they are not extracting RNA and I'm afraid that these protocols will affect the RNA quality. Therefore, when I found your HistoReveal I got a bit curious... Just tell me if there is any more information that you need!

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      Answer

      Thank you for confirming the details. The HistoReveal ab103720 use in IHC is based on enzymatic antigen retrieval method. As there is no step of heating the tissue sections so probably this could be product as per your requirements. However I cannot be 100% sure because we have never tried this product in our own lab in similar kind of experiment so we will not be able to share any data regarding this. Please be advised that the optimization of experiment has to be done by the end user. I would also suggest searching the literature about the common method other scientist are using. I have done quick search which I am happy to share; http://jhc.sagepub.com/content/49/8/931.full.pdf+html Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K (1999) Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res 27:4436–4443 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC148555/pdf/270e12.pdf I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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      Question

      Hi! I'm going to use FFPE sections for laser-capture microdissection of cells for RNA extraction and qPCR. I'm planning to visualize the cells of interest with IF and has been looking around for a suitable antigen retrieval protocol that does not harm the RNA. Would you say that HistoReveal is suitable for this application or if you have any better suggestion? Best Regards

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      Answer

      Thank you for contacting us. Could you specify which target you would be interested in seeing with antibodies? I don’t think the antigen retrieval method will affect the target detection in IHC-IF. However if you are interested in extracting RNA from FFPE tissue sections then the procedure will be different. Because formaldehyde affects nucleic acids by forming adducts with exocyclic nitrogen atoms in nucleotides. This immobilization of DNA and RNA is attributed to trapping of the long nucleic acid molecules in networks of associated basic protein molecules, which are cross-linked by methylene bridges. Could you also provide more information about the experiment you are planning to do? I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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