Cell Membrane Staining (WGA)¶
Morphology staining performed after signal readout (PRISM imaging or SPRINTseq sequencing). WGA labels cell membranes and cytoplasm for cell boundary segmentation.
- Applicable samples: Any permeabilised section after fluorescent signal readout, on-slide or in flow cell.
- Version: v1.0
- Last modified: 2026-08-04
Materials
Stock preparation: WGA-488 Stock · DAPI Stock — prepare and aliquot these ahead of time. Ordering: Bill of Materials → Morphology.
Specificity: N-acetylglucosamine (GlcNAc) and sialic acid on cell membranes. For the fluorophore, see Conjugate choice — it determines both the imaging channel and the flow-cell carryover risk.
Clarify the stock before every use
Warm the aliquot at 37°C briefly, then centrifuge 10,000g for 1 min and use the supernatant only. Precipitated conjugate is the main cause of speckled background and of clogged flow-cell tubing. See WGA-488 Stock.
Conjugate choice¶
| WGA-AF488 (green) | WGA-XFD647 / AF647 (far-red) | |
|---|---|---|
| Ex/Em | 495 / 519 nm — FITC channel | ~650 / 671 nm — Cy5 channel |
| Dye class | Sulfonated fluorescein (xanthene) | Cyanine |
| Surface behaviour | Two sulfonates, strongly hydrophilic; little nonspecific binding | Sulfonated but retains a large hydrophobic polymethine core; adsorbs to tubing and plastics, and self-stacks |
| Flow-cell carryover risk | Low | High — see Line cleaning |
Choose the conjugate that keeps WGA out of the channels the readout uses. In a two-colour SBS readout the sequencing channels are cy3 and cy5, so the green conjugate is out-of-band: any residue it leaves in the fluidics shows up in a channel that is not used for base calling. A far-red conjugate shares the cy5 channel, so its carryover appears directly as sequencing background on the next run.
On an automated system, prefer the green conjugate
Both the dye chemistry and the channel assignment point the same way: cyanine conjugates stick to the flow path far more than sulfonated fluoresceins, and what they leave behind lands in a sequencing channel. Use far-red only when something else in the panel needs the green channel, and step up the line cleaning if you do.
Manual Staining (Off Flow Cell)¶
Typical workflow: after signal readout → remove from Flow Cell → WGA staining → mount.
- Wash: PBS, 2 x 2--3 min.
- Prepare working solution: Dilute stock to 5--10 μg/ml in PBS.
- Stain: Apply working solution, incubate RT for 15 min, protected from light.
- Wash: PBS, 2 x 2--3 min.
- Mount: PBS or anti-fade mountant. Cover with coverslip, avoid bubbles.
- Image: FITC channel (488 nm excitation).
Automated Staining (In Flow Cell)¶
Can be combined with DAPI as a single staining mix:
- DAPI/WGA Mix: 10 ng/ml DAPI + 5--10 μg/ml WGA in PBS. (DAPI at 5 μg/ml bleeds into the 480 nm channel and will contaminate the WGA/FITC signal — see DAPI Stock.)
- Pump into Flow Cell, stop flow, incubate RT 15 min.
Line cleaning (critical)¶
WGA is the last step of a run, so whatever it leaves on the shared flow path stays there until the next run, where the following reagents desorb it onto fresh tissue. The failure mode is cross-run carryover, not same-run background.
Immediately after imaging: flush with 4X SSC (or 0.5 M NaCl) 1 min, then PBS-T (0.1% Tween) 1 min. High salt screens the electrostatic attraction between WGA (basic, pI ≈ 8.5, so cationic at neutral pH) and the negatively charged tubing and glass; the nonionic detergent addresses hydrophobic adsorption.
The immediate rinse alone does not prevent cross-run carryover
Neither salt nor detergent destroys WGA — they relocate an intact, still binding-competent lectin. WGA resists detergent because each monomer carries 16 disulfide bonds (four hevein domains × four), so it does not unfold in SDS or Tween without a reducing agent. Three practices matter:
- Flush both flow paths. Where the instrument has a flow-cell bypass, a bypass-only flush never touches the segment between the flow-cell inlet valve and the chip — the one segment that can carry contaminant onto the next sample. Run the rinse through the flow cell as well as the bypass.
- Decontaminate periodically with alkali. A soak in 0.1 M NaOH hydrolyses the protein instead of moving it. Optionally add 10 mM DTT + 1 mM EDTA, mixed fresh — DTT breaks the disulfides, and additionally bleaches cyanine dyes by adding to the polymethine chain, so it attacks a far-red conjugate's fluorophore directly. At pH 13 DTT is fully thiolate, which is both why it reduces quickly and why it autoxidises within hours; it will be spent well before a long soak ends.
- Never follow a detergent wash with alcohol. At fixative concentrations alcohol precipitates residual protein onto the tubing wall, leaving it harder to remove than before. If a solvent step is wanted, place it after the protein has been hydrolysed and keep it dilute (≤20 % isopropanol), which also improves wetting of poorly-swept fitting dead volume.
If cleaning does not fix it, stop escalating the wash
Contamination that survives a reducing alkaline soak is usually not a chemistry problem: either the solution never reaches the reservoir (unswept dead volume at a fitting — replace the short tubing run instead), or the background is not WGA at all. Incorporation mixes can bind tissue membranes nonspecifically and look identical. The test that separates them is an abbreviated readout cycle on a fresh flow cell with no tissue.
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.
Changelog¶
- v1.0 -- 2026-08-04 -- First versioned release. Line cleaning promoted to its own section and expanded: the 4X SSC → PBS-T rinse is retained, with the mechanism it works by (charge screening against a cationic lectin) now stated, but it is no longer presented as sufficient on its own — WGA's 16 disulfides per monomer make it detergent-resistant, so a salt/detergent rinse relocates an intact lectin rather than destroying it. Added the requirement to flush the through-flow-cell path and not only the bypass, periodic 0.1 M NaOH ± DTT/EDTA decontamination, a caution against following detergent with alcohol, and a stop rule for when further washing is the wrong response. New Conjugate choice section covering far-red (XFD647/AF647) alongside AF488, including the much higher carryover risk of cyanine conjugates and the fact that far-red residue lands in the cy5 sequencing channel. Title generalised from "WGA-Alexa Fluor 488" to "WGA".