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When deciding on an antibody, the following points should be considered to increase the likelihood of high specificity and low cross-reaction and a successful staining; further optimization will then help to achieve a reliable signal: Consulting literature and antibody comparison resources, checking in-house validation data of the antibody seller, searching for independent validation data, and consulting the original manufacturer for technical service and protocol support.
The initial choice of the primary antibody can affect the whole outcome of the experiment. Thus, it is important to search for an antibody that is well validated to increase the chance of a successful experiment. Most importantly, it is helpful to purchase the antibody from the original manufacturer and not from a vendor, as only the original manufacturer has access to all validation data and can assist with superior technical support.
|Main questions to ask when choosing a primary antibody supplier|
|1||How many times has the antibody been cited?|
|2||How has the antibody been validated?|
|3||Has the application of interest already been tested?|
|4||Who is the original manufacturer?|
|5||Is all the data and information about the antibody publicly available?|
|6||What is the vendor’s refund policy, delivery time, and stock availability?|
|7||What is the price?|
Benefits of polyclonal antibodies in IHC
Drawbacks of polyclonal antibodies in IHC
Benefits of monoclonal antibodies in IHC
Drawbacks of monoclonal antibodies in IHC
General steps on optimizing conditions for a primary antibody in IHC
Polyclonal primary antibodies are mainly IgG isotypes. Primary monoclonal antibodies are occasionally of a different isotype and therefore need an isotype-specific antibody.
Secondary antibodies can go through an additional purification step to reduce potential cross-reactions with other species. Therefore, the secondary antibody solution is passed over different columns containing sera proteins of different species to filter out the non-specific secondary antibody.
High background staining can be due to the presence of Fc receptors in special tissue or cells.