What are the factors that affect the outcome of immunohistochemical studies?

Factors that affect the outcome of immunohistochemical studies include temperature, pH, cold ischemia time, ionic conditions, duration of incubation, storage time, fixation and tissue processing, and number of epitopes sites per cell. Shorter ischemia times are generally preferred to minimize tissue damage. Alterations from the optimal pH range can lead to denaturation of proteins and loss of antigenicity. High salt concentrations or the presence of chaotropic agents can disrupt protein structures. Tissues with high expression levels of the target antigen may exhibit stronger staining signals, whereas tissues with low antigen density may require longer incubation times. Insufficient incubation times may result in weak or incomplete staining, whereas prolonged incubation times can increase background staining. Variations in temperature can affect the kinetics of antibody-antigen binding. Inadequate fixation or tissue processing may lead to antigen degradation. An effective fixative should adequately preserve cellular structures by hardening the tissue while simultaneously inhibiting enzymatic and chemical degradation processes. Proper paraffin embedding ensures tissue integrity and provides support during sectioning. The length of time tissue samples are stored after fixation and embedding can impact antigen preservation and staining quality.