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WGA-Alexa 488 Staining Protocol

Product Information

  • Product: Alexa Fluor 488 labeled Wheat Germ Agglutinin (WGA-488)
  • Catalog: S33667-1mg
  • Storage: -20°C, protected from light, stable for at least 1 year
  • Fluorescence: Ex/Em = 495/519 nm (green)
  • Specificity: N-acetylglucosamine (GlcNAc) and sialic acid

1. Stock Solution Preparation

  1. Warm up: Bring WGA-488 (1 mg) to room temperature for at least 30 min.
  2. Centrifuge: Low-speed spin for 2-3 min to collect condensation.
  3. Dissolve: Add 500 μL sterile ddH₂O to prepare 2 mg/mL stock solution.
  4. Aliquot & store:
    • Short-term: 2-8°C
    • Long-term: Aliquot 10-20 μL/tube, store at -20°C protected from light
    • Avoid repeated freeze-thaw cycles; stable for at least 1 month

Before Use

  • Remove precipitate: If stored long-term, warm at 37°C for a few minutes.
  • Centrifuge: 10,000g for 1 min, use supernatant only.
  • This reduces non-specific background and prevents clogging.

2. Usage (Post-RCA Frozen Sections)

Applicable Conditions

  • Sample type: Fixed and RCA-amplified frozen tissue sections
  • Section thickness: 10 μm
  • Pre-treatment: Already permeabilized (Pepsin, Methanol)
  • Workflow: After RCA → in-situ sequencing → DAPI staining → remove from Flow Cell → WGA staining → re-mount

Working Solution

  • Dilute 2 mg/mL stock in PBS to working concentration
  • Working concentration: 5-10 μg/mL (recommended starting point)
  • Dilution factor: 1:400 to 1:200
  • Buffer: PBS (recommended). Avoid cell culture media (increases non-cellular background)
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

Manual Staining Steps

  1. Remove slide from Flow Cell carefully, keeping section intact.
  2. Wash: PBS, 2 x 2-3 min to remove residual sequencing reagents.
  3. Prepare working solution: Warm stock at 37°C briefly → centrifuge (10,000g, 1 min) → take supernatant → dilute in PBS.
  4. Stain: Apply working solution to fully cover the tissue. Incubate RT, 15 min, protected from light.
  5. Wash: PBS, 2 x 2-3 min to remove unbound WGA.
  6. Mount: Apply PBS or anti-fade mountant (e.g., ProLong Gold). Cover with coverslip, avoid bubbles.
  7. Image: Excitation 488 nm (FITC channel), Emission 519 nm.

Notes

  • Since sample is permeabilized, WGA enters cell interior — labels both membrane and cytoplasm (especially Golgi region). This is expected and does not affect cell segmentation.
  • If background is too high, reduce concentration. If signal is weak, increase up to 20 μg/mL max.
  • Always centrifuge stock before use to remove precipitate.
  • If re-mounting in Flow Cell is needed, use PBS mounting (not anti-fade mountant).

Application

  • Cell segmentation mask for spatial transcriptomics (PRISM, SPRINTseq)
  • Compatible with Cellpose, Baysor, and similar segmentation algorithms
  • Expected pattern: DAPI (nuclear, empty), WGA (cytoplasm/membrane, bright) — "honeycomb" or "solid fill" structure for boundary detection