![]() ![]() A strong background signal suggests interference from endogenous peroxidases or phosphatases. Incubate a test tissue sample with the detection substrate alone for a length of time equal to that of the antibody incubation. See also the additional notes sections at the bottom of this page for more information. The following points are provided to help identify the cause of high background staining, which results in a poor signal-to-noise ratio. The sections were counterstained with hematoxylin and dehydrated with ethanol and xylene prior to mounting. Detection was performed using an HRP-conjugated secondary antibody followed by chromogenic detection using DAB as the substrate. Tissues were washed extensively in PBS buffer containing 0.05% (v/v) Tween-20 (PBST). After HIER, tissues were blocked in 3% H2O2 in methanol for 15 minutes at room temperature, washed with distilled H2O and PBS, and then probed overnight at 4☌ in a humid environment with an Invitrogen Connexin 43 monoclonal antibody (Cat. To expose target proteins, heat-induced epitope retrieval (HIER) was performed using 10 mM sodium citrate (pH 6.0), followed by heating in a microwave for 8 to 15 minutes. Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) cancer tissue. Analysis was performed to compare Connexin 43 membrane staining in FFPE sections of human lung adenocarcinoma (right) compared to a negative control without primary antibody (left). The following image provides an example of IHC staining. ![]()
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