Abstract: Disclosed are compositions, methods, systems, and kits for the detection of markers of interest, which may allow for sequential multiplexing. In some cases, the methods comprise sequential staining of biological samples with detection sets and removal of bound detection agents which may allow for sequential staining with additional detection agents.

Abstract: Provided herein are immunofluorescence assays that do not use an intensely colored lipophilic dye, and that result in a significant reduction of fluorescence due to aldehyde fixation, collagen, elastin, and/or red blood cells. In certain embodiments of the present invention, a two-step treatment of the tissue is contemplated that leads to the incorporation of reduced heteropoly acids or salts thereof (e.g., phosphomolybdate species) in the tissue. This reagent binds tightly to hydrophilic regions of the tissue and effectively quenches the fluorescence in those regions. In doing so, the overall signal-to-noise ratio for the immunofluorescence assay is increased.

Abstract: The present disclosure generally relates to methods for detecting one or more analytes in a sample using immunoassays. In some embodiments, the disclosure provides methods that include quenching the free secondary antibody with a fractionated serum composition. In some other aspects, the disclosure provides systems and kits suitable for carrying out the foregoing methods and any embodiments thereof.