Glysite™ Scout Glycan Screening Kit, Immunofluorescence 649

Virtually any tissue type and species are suitable for use with a Glysite Scout Glycan Screening Kit, Immunofluorescence, as glycans are ubiquitous in nature and in life. The kit has been validated on human and mouse tissue—including FFPE tissue, across a range of organs, including colon, lung, spleen, kidney, liver, pancreas, testes, heart, bladder, and uterus.

Each of the three Glysite Scout Glycan Screening Kits, Immunofluorescence contain the same lectins but with a different fluorescent streptavidin detection reagent, offering a choice of Dylight™ 488 (GSK-3000), Dylight™ 594 (GSK-2000), or Dylight™ 649 (GSK-1000). This allows users to choose the kit which best matches the capabilities of their fluorescent microscope and also provides flexibility for multiplexing with other fluorescent detection reagents they might already be using. 

VECTASHIELD Vibrance is an antifade medium for immunofluorescence applications with setting properties, which means the coverslip is immobilized after curing and does not require sealing. Additionally, slides can be stored at room temperature, enabling extended archiving time. VECTASHIELD PLUS does not have setting properties and remains molten, and slides must be stored at 2–8°C. Both VECTASHIELD Vibrance and VECTASHIELD PLUS have superior antifade/anti-photobleaching properties across the spectrum and are compatible with commonly used fluorophores. 

The need for an antigen retrieval step in a staining protocol depends on multiple variables, including the target glycan and its location in the specimen, the glycan-binder, the type of tissue, and the method and length of fixation. Investigators should run controls with and without antigen retrieval to determine if this step is needed for their specimen. 

Tissue components such as red blood cells, elastin, and collagen are strongly fluorescent, particularly in FFPE sections, making it difficult to discern between relevant signal and background. Formalin fixation tends to induce a significant amount of fluorescence to a specimen. The Vector TrueVIEW Autofluorescence Quenching Kit provides a dramatic reduction of autofluorescence, allowing a clear view of the desired signal, so it was utilized as needed on the specimens represented in the images. 

If co-labeling with a primary antibody, users should optimize each protocol separately to achieve maximum signal to noise ratios. Once this is complete, the two protocols can be combined, with the Glysite Scout Glycan Screening Kit protocol applied first, followed by the antibody staining protocol. Users should perform the proper controls to demonstrate that the reagents in each protocol are not cross-reacting with each other. 

The Carbo-Free Blocking Solution included in the Glysite Scout Glycan Screening Kit is virtually free of glycoproteins or other glycosylated molecules. This is critical since these kits detect glycans and would therefore bind to blocking reagents which contained glycosylated components. Users should not use other blocking solutions (e.g., milk derivatives, sera) as it may lead to high background. 

Each Glysite Scout Glycan Screening Kit contains enough reagents for approximately 50 tissue samples per lectin, or up to 400 samples if using one lectin per tissue sample (8 lectins x 50 tissue samples). 

The lectins were selected based upon their broad applicability and specificity to frequently cited glycan targets from literature sources, spanning fields of Cancer, Immunology, Metabolism, Inflammation, Virology, and Tumor Microenvironment, among others. They enable detection of all the major glycan motifs for the evaluation of glycan distribution in a target specimen.