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Nuclear Staining (DAPI)

  • Applicable samples: Any section after signal readout (PRISM imaging or SPRINTseq sequencing)
  • Version: v1.0
  • Last modified: 2026-08-18

Morphology staining performed after signal readout (PRISM imaging or SPRINTseq sequencing). DAPI labels cell nuclei for segmentation and structural context.

Materials

Stock preparation: DAPI Stock — 5 mg/ml stock and the serial dilution to working concentration. Ordering: Bill of Materials → Morphology.

Reagents

Reagent Stock Working Conc. Dilution from stock Notes
DAPI 5 mg/ml 10 ng/ml — multiplexed panels 1:500,000 Preferred default. No bleed into the 480 nm channel
DAPI 5 mg/ml 5 μg/ml — DAPI-only imaging 1:1000 Legacy concentration; bleeds into 480 nm

Nuclear DNA intercalator; Ex/Em 358/461 nm.

Use 10 ng/ml unless you are imaging DAPI alone

The long-standing 1:1000 dilution (5 μg/ml) is far more dye than nuclear staining needs. At that concentration DAPI crosstalks into the 480 nm channel, which was found in-house while setting up six-colour imaging -- it eats a channel you need for signal.

10 ng/ml stains nuclei perfectly adequately and does not crosstalk. Use it for any panel that images more than DAPI. The 5 μg/ml condition is retained only for DAPI-only morphology scans, where the bleed costs nothing.

Dilution is a two-step serial (1:1000, then 1:500) — see DAPI Stock Preparation. Make the final dilution fresh.

Manual Staining (Off Flow Cell)

  1. Wash: PBS, 2 x 2 min.
  2. Stain: Apply DAPI (10 ng/ml in PBS; 5 μg/ml only for DAPI-only imaging), incubate RT for 10--15 min, protected from light.
  3. Wash: PBS, 2 x 2 min.
  4. Image: DAPI channel (405 nm excitation).

Automated Staining (In Flow Cell)

For SPRINTseq workflows using a flow cell and pump system:

  1. Wash (pre-clean): Pump PBS-T (0.05% Tween), flush 2 min to remove residual sequencing reagents.
  2. Stain: Pump DAPI (10 ng/ml in PBS) until Flow Cell is filled. Stop flow. Incubate RT for 15 min.
  3. Wash (post-clean): Pump PBS, flush 2 min.
  4. Image: Full slide scan in DAPI channel.

See also: Cell Membrane Staining (WGA) | H&E Staining

Changelog

  • v1.0 -- 2026-08-18 -- First versioned release of this page. The staining procedure is unchanged (page content dates to 2026-07-30); the metadata block and this changelog were added so the page conforms to the house format and appears in the protocol index. See docs/CONTRIBUTING.md for the format.

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