Glysite™ Explorer in situ PLA Glycan Detection Kit & Glysite™ Explorer Lectins

The Glysite Explorer Kit allows the visualization of a glycan and a protein when they are within 40nm of each other.

Customers will need to purchase a Glysite Explorer Lectin, and for additional reagent requirements, please see the User Guide.

• The Glysite Explorer kit has been validated on human specimens.
• Other species of tissues should also be compatible, but the primary antibody species should be selected to avoid endogenous IgG detection.

The Glysite Explorer Kit has been validated on formalin-fixed, paraffin-embedded (FFPE) tissues, FFPE cells, and fixed cells. The kit is not recommended for use on frozen tissue specimens at this time.

• The concentrations and incubation times in the Glysite Explorer Kit have been optimized for 4-6 µm sections. We have not evaluated the performance of this kit on thick sections or free-floating sections.
• For users who want to stain thick sections, the protocol will need to be modified.

• The Glysite Explorer Kit includes two separate secondary antibody reagents: one anti-mouse and one anti-rabbit. This allows users to pair either a mouse or a rabbit primary antibody with their chosen Glysite Explorer Lectin, depending on the needs of the experiment.
• We do not recommend the use of a primary antibody from the same species as the tissue being studied—for example, do not use a mouse primary on mouse tissue—to minimize nonspecific binding and reduce the risk of false-positive signals from endogenous IgG.

• Multiplexing with the Glysite Explorer Kit is generally not recommended.
• This kit has not been validated to perform multiplexing. In some cases, it may be possible if detection reagents do not cross-react and the proper controls are included.

• Positive controls
  • Characterize the expression of the protein of interest in your specimen using standard Immunohistochemistry (IHC), Immunofluorescence (IF) or other workflows.
    • Determine the optimal dilution for the primary antibody in a standard IHC assay as this will be the recommended starting dilution for the isPLA assay.
  • Characterize the expression of the glycan of interest in your specimen using standard Immunohistochemistry (IHC), Immunofluorescence (IF) or other workflows.
•  Negative control:
  • Omit the Glysite Explorer Lectin and/or primary antibody from the workflow to demonstrate whether non-specific binding or background staining is present.

Reagent volumes should be determined by the researcher to align with the dimensions of their specimens. Volumes sufficient to cover the specimens should be applied, and samples should not be allowed to dry out during the incubation steps.

The number of specimens that can be stained will vary depending on specimen size. The Glysite Explorer Kit provides 5 mL of working solution. If using 50-100µL of reagent per slide, there is sufficient volume to stain 50-100 specimens.

Slides stained with the Glysite Explorer Kit can be mounted with either an aqueous or a non-aqueous mounting medium. For non-aqueous mounting, we recommend using VectaMount® PT Permanent Mounting Medium (H-5600-60) or VectaMount® Express Mounting Medium (H-5700-60). For aqueous mounting, we recommend using VectaMount® AQ Aqueous Mounting Medium (H-5501-60).

• The Glysite Explorer Kit generates distinct, round signals called puncta at the site of proximity. These are best visualized using at least a 20x objective, and sometimes a 40x may be required, especially when epitope density is low.
• If the counterstain is too dark, it can obscure isPLA signals. Hematoxylin QS incubation should be optimized for each specimen to ensure positive signals are not masked. Even 1 second of staining is often sufficient, if performed at all.
• The overall appearance will vary by specimen and proximal target abundance.

The average run-time will depend on the duration of the primary antibody incubation. If your primary antibody incubation is 30 minutes, the total run time is approximately 8 hours.

If the assay cannot be completed within a single day, the tissue sections can be stored overnight at 4°C in Protein Diluent after incubation of the Lectin Solution in step 13. Do not allow the sample to dry out.

The kit is supplied with ImmPACT DAB, Vector’s most sensitive peroxidase substrate. Other peroxidase substrates can be substituted, and the Glysite Explorer Kit has been validated with ImmPACT® NovaRed (Catalog Number: SK-4805), ImmPACT® VIP (SK-4605), and ImmPACT® SG (SK-4705). However, not all peroxidase substrates are equivalent in sensitivity and staining quality and should be optimized to achieve desired results.

No, the Glysite Explorer Kit is not currently available with a fluorescent readout.

• The Glysite Explorer Kit is supplied in two boxes, and the labels indicate the proper storage conditions. Box 1 should be stored at 4°C. Box 2 should be stored at -20°C.
• The Glysite Explorer Lectins should be stored at 4°C.