SOP: Wheat Germ Agglutinin (WGA) staining combined with DAPI- Thermofisher

Wheat Germ Agglutinin (WGA) staining combined with DAPI  is commonly used in cell biology and microscopy to visualize and study the cell surface and nucleus, providing insights into cell morphology, and viability under various physiological and non, conditions.

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Utility: WGA, is a lectin that binds specifically to N-acetylglucosamine and sialic acid residues present on the surface of cells.  Thanks to this property, it is widely used for cell membrane and extracellular component visualization and imaging applications.  Usually, this is applied together with DAPI, which binds to the DNA, and, so this co-staining allows for cell membrane and nuclei observation, especially for imaging studies to assess morphology, cell viability, and spatial location and connection. 

How It Works: WGA conjugated to a fluorescent dye (multiple fluorophores available) binds to glycoprotein components on the cell surface. The specific concentration and incubation times ensure optimal staining while minimizing non-specific binding. DAPI penetrates the cell and binds to DNA, providing a strong fluorescent signal from the nuclei. The short incubation time prevents excessive staining that can lead to high background fluorescence.

Watch Out For:

• Ensure the WGA solution is clear of aggregates by centrifugation to avoid non-specific staining. Always prepare fresh solution at the moment if the staining.
• Make sure to avoid excessive cell exposure and fixation with PFA, reducing WGA binding efficiency due to possible masking of epitopes.
• High concentrations of DAPI can cause overshadowing other signals.

Additional Tips:

• Follow detailed instructions for time and temperature incubation to ensure staining specificity and efficiency. 
• Optional depending on the selected chamber slide. Important to use antifade mounting media to preserve fluorescence signal. 
• Keep the slides moist throughout the staining process to preserve cell morphology.
• Use a needle or tweezers to guide the coverslip gently onto the slide to avoid trapping air bubbles and cell distortion.
• If appropriately stained and stored in the dark, the fluorescence could be maintained for up to 4 days).

Step Preparations:

5 ug/ml WGA solution

              -Centrifuge WGA vial to remove protein aggregates

                             -Dilute 1 mg/ml (5 ul) into 995 ul of HBSS

              4 % PFA

                             -Dilute 1 ml (16% stock solution) into 3 ml PBS

              DAPI

-Take 1 ul of DAPI stock (14.3 mM) and add it to 1 mL of PBS to create a 300 nM working solution solution.

1. Aspirate media and briefly wash chambered slide in PBS
2. Fix in 4% PFA for 10 minutes at room temperature
3. Wash by aspirating PFA and carefully adding PBS to the chamber. 
4. Repeat Step 3.
5. Incubate slide with 5 ug/ml WGA solution for 10 min at 37 C
6. Wash twice in PBS.
7. Incubate the slide with DAPI solution for 1-5 min. 
8. Aspirate DAPI solution and wash once in PBS.
9. Remove the walls of slide carefully and add sufficient drops of mounting media (you can also use ready-to-use chamber slides where you do not need to use the mounting media).
10. Carefully place a coverslip on the slide while avoiding any bubbles. 
11. After overnight incubation, store at 4 C.

Representative image: