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Cell Membrane Staining (WGA-Alexa Fluor 488)

Morphology staining performed after signal readout (PRISM imaging or SPRINTseq sequencing). WGA labels cell membranes and cytoplasm for cell boundary segmentation.

Product Information

Property Value
Product Wheat Germ Agglutinin, Alexa Fluor 488 conjugate
Catalog Thermo Scientific W11261 (or S33667)
Fluorescence Ex/Em = 495/519 nm (green, FITC channel)
Specificity N-acetylglucosamine (GlcNAc) and sialic acid on cell membranes
Storage -20°C, protected from light

Stock Solution (2 mg/ml)

  1. Bring WGA-488 (1 mg) to room temperature for ≥30 min.
  2. Centrifuge briefly to collect condensation.
  3. Dissolve in 500 μl sterile ddH₂O → 2 mg/ml stock.
  4. Aliquot 10--20 μl/tube, store at -20°C protected from light.

Before use

Warm aliquot at 37°C briefly, then centrifuge 10,000g for 1 min. Use supernatant only (removes precipitate that causes background).

Manual Staining (Off Flow Cell)

Typical workflow: after signal readout → remove from Flow Cell → WGA staining → mount.

  1. Wash: PBS, 2 x 2--3 min.
  2. Prepare working solution: Dilute stock to 5--10 μg/ml in PBS. 
    5 μg/ml:  2.5 μl stock (2 mg/ml) + 997.5 μl PBS
10 μg/ml: 5 μl stock (2 mg/ml) + 995 μl PBS
  3. Stain: Apply working solution, incubate RT for 15 min, protected from light.
  4. Wash: PBS, 2 x 2--3 min.
  5. Mount: PBS or anti-fade mountant. Cover with coverslip, avoid bubbles.
  6. Image: FITC channel (488 nm excitation).

Automated Staining (In Flow Cell)

Can be combined with DAPI as a single staining mix:

  • DAPI/WGA Mix: 5 μg/ml DAPI + 5--10 μg/ml WGA-488 in PBS.
  • Pump into Flow Cell, stop flow, incubate RT 15 min.
  • Line cleaning (critical): After imaging, flush with 4X SSC (or 0.5 M NaCl) 1 min, then PBS-T (0.1% Tween) 1 min. This prevents WGA from adhering to tubing.

Notes

  • Since tissue is permeabilized, WGA enters cell interior -- labels both membrane and cytoplasm (especially Golgi). This is expected and useful for segmentation.
  • If background is too high, reduce concentration. If signal is weak, increase up to 20 μg/ml.
  • Compatible with Cellpose, Baysor, and similar segmentation algorithms.
  • Expected pattern: DAPI (nuclear) + WGA (cytoplasm/membrane) → "honeycomb" structure for boundary detection.