Reasons Why IHC Optimization Tips Staining Is Important?

Posted by Immuno Staining on July 12th, 2019

Immunohistochemistry widely known as IHC is the most popular technique that uses antibodies to visualise the particular type of antigens within stored and preserved tissue sections. Compared to Western blotting practices that involve analysis of bulk samples like tissue Lysates, IHC provides the most valuable and critical information regarding relative expression levels in the context of tissue anatomy and antigen localisation. IHC is a critical technique which is used in both life science and clinical research laboratories by combining the power of the specific type of microscope and antibodies to reveal both abundance and location of a particular protein in a tissue section.

In theory, the protocol is very straight forward: A thinly sliced formalin-fixed or flash-frozen and paraffin-embedded tissue sample are placed on a microscope slide and it is incubated with antibodies specific to the protein of interest. This process is followed by performing signal detection by using a fluorescent dye or a chromogenic substrate. The signal detection is observed microscopically in particular areas where the antibody is bound to the protein of interest. But in reality, optimising the necessary protocol steps for an Immunohistochemistry (IHC) assay can be labour intensive and involves managing several key parameters. Even though a standard protocol sometimes may work very well with particular antibodies, but the same type of protocol may not work perfectly well with all antibodies particularly those which may require protocol modifications in order to generate correct results.

In general, compared to structurally complex and thicker tissue sections, the fixation times and strengths are considerably shorter for cells. And in order to ensure free access of the antibody to its antigen then it is important that the cells must be permeabilized and fixed. Boster antibody and ELISA experts provide IHC Optimization Tips Staining for selecting the best fixative for your sample. The fixation of IHC protocol prevents necrosis and autolysis of excised tissues by preserving their antigenicity and their morphology as well as increasing their resistance to processing. As different fixatives are more optimal for some antigens and antibodies and others, so it is important to select the right fixation method.

Once you have selected the right fixation method for your fixative then the very next step is to apply it to your sample protocol. In general, there are two common ways to fix tissue i.e. perfusion and immersion. Immersion is the most ideal and common method suitable for excised tissue samples and cell cultures and the amount of time taken for this method varies depending upon the type and size of tissue being fixed. Boster is the one-stop solution for all the Protocols, optimisation tips, troubleshooting guides and more for IHC Fixation Protocol Staining. For more details to know about Boster antibody and ELISA expert please visit our website here: https://immunostaining.info/