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Autofluorescence Quenching

  • Applicable samples: Any RCA'd section going into fluorescence readout. Sample-dependent -- FFPE almost always needs it; high-lipofuscin FF tissue (e.g. brain, aged tissue) benefits; low-autofluorescence FF often does not.
  • Version: v1.0
  • Last modified: 2026-07-13

When and why

Autofluorescence (lipofuscin, aldehyde-fixation products, extracellular matrix) raises background and can swamp rolling-circle signal. Quenching is optional and sample-dependent. Run it after RCA (post-amplification fix) -- never during library construction, where the post-amplification fix would re-introduce aldehydes and where later washes strip any masking dye. The same choice applies whether the downstream readout is PRISM or SPRINTseq.

Before or after fluorescent staining -- a trade-off to test

Within the post-RCA window there is one placement decision left: quench before or after the fluorescent probe staining.

Applying the quench after staining generally gives the best background suppression -- nothing downstream washes the quencher back out, and you are quenching against the exact tissue state you will image. But it can attenuate the true signal too, since no quencher distinguishes rolling-circle fluorophore from autofluorescence. The net gain depends on tissue, panel and fluorophore set: test both orders on control sections before committing a sample.

As a starting point, the masking options (Sudan Black B, TrueBlack) benefit most from going after staining -- they are the ones later washes remove. The destructive option (NaBH₄ + photobleaching) is permanent either way, so it is usually placed before staining unless autofluorescence is severe.

Where this fits in the workflow

Upstream: RCA Protocol + post-amplification fix. This step (optional): quench autofluorescence. Downstream: signal readout -- PRISM Imaging or SPRINTseq Sequencing.

Pick one quencher by sample type. All three are optional and should be tested on a control section first -- each can reduce true signal if over-applied.

Option A - NaBH₄ + photobleaching (FFPE)

Goal: Reduce aldehyde-induced autofluorescence on FFPE; the standard choice for archival FFPE sections. The chemical quench and the physical (light) quench run concurrently, not in sequence.

  • Incubate: 1% (wt/vol) NaBH₄ (in 1X PBS) for 30 min at RT, under strong light (LED) illumination for simultaneous photobleaching.
  • Wash: PBS-Tween 0.05% 3 x 2 min.

Split the incubation to keep the reagent fresh

NaBH₄ is consumed and gases off during the incubation. An equivalent and often better-controlled schedule is 6 min x 5 rounds with fresh solution each round, rather than one continuous 30 min block.

Photobleaching alone

Strong-light photobleaching adds no reagent and masks nothing, so it can be extended when autofluorescence is severe (overnight bleaching has been used). Unlike the masking options below, it costs only time - not signal.

NaBH₄ safety

Prepare 10 ml of 1% (wt/vol) NaBH₄ in 1X PBS on ice, freshly each time. Use extreme caution when weighing; accidental introduction of liquid into the stock bottle can cause an explosive reaction. Gas bubbles form continuously in the chamber -- aspirate and replenish with fresh solution every 1-2 min during the incubation.

Option B - Sudan Black B (FF, general)

Goal: General lipofuscin / autofluorescence suppression for FF sections.

Stock: 0.1% (wt/vol) Sudan Black B in 70% EtOH (e.g. 10 mg in 10 ml). Shake at RT for days, filter before use.

  • Incubate: 0.1% Sudan Black B (filtered) at RT for 10 min.
  • Wash: 70% EtOH 10 x 3 min, then PBS-Tween 0.05% 2 x 1 min.

Warning

Sudan Black B may reduce signal intensity. Test on control samples first.

Option C - TrueBlack / TLAQ (high-lipofuscin)

Goal: Strong lipofuscin quench for high-autofluorescence tissue (e.g. human brain), as used in HybISS-style readout.1

Reagent: Biotium TrueBlack Lipofuscin Autofluorescence Quencher, cat. 23007 (20X in DMF, 1 mL, ~100-200 sections). An aqueous variant, TrueBlack Plus cat. 23014 (40X in DMSO, diluted 1:40 in PBS, 5-10 min RT), avoids the ethanol step below and is worth preferring where ethanol is a problem.

  • Prepare the stock: heat the 20X vial to 70 °C for 5 min before diluting - otherwise it deposits precipitate on the section. Centrifuge before opening.
  • Prepare working TLAQ: dilute 20X TrueBlack (TLAQ) stock 1:20 in 70% ethanol to a 1X working solution. Use 100-200 μl per section.
  • Incubate: apply 1X TLAQ, RT 30 s - 1 min (brain: 30 s - 2 min tested; optimize per tissue).
  • Wash: immediately with PBS, then 2 more times (3 total).

Rinse in PBS first - SSC precipitates it

Biotium documents that TrueBlack "may precipitate out due to the high salt SSC wash buffers used", and recommends rinsing sections in PBS immediately before treatment. Since the hybridization and stringency washes upstream are SSC-based, always PBS-rinse before applying.

Protecting a TrueBlack quench

After TrueBlack, avoid detergents and ethanol (they wash it out), avoid high formamide, and keep the section in the dark (light-sensitive). In a cyclic readout that uses a formamide strip between rounds, that strip removes TrueBlack -- re-apply each cycle as needed. TrueBlack may alternatively be applied after the readout-probe hybridization.

References

  1. Gyllborg D, Nilsson M. HybISS: Hybridization-based In Situ Sequencing. protocols.io (2020). DOI: 10.17504/protocols.io.xy4fpyw. — source for the TrueBlack/TLAQ step (Option C).
  2. Biotium, TrueBlack Lipofuscin Autofluorescence Quencher product information. — Option C reagent.

NaBH₄ (Option A) and Sudan Black B (Option B) are standard autofluorescence-quenching reagents in fluorescence in situ work.

  1. Gyllborg D, Nilsson M. HybISS: Hybridization-based In Situ Sequencing. protocols.io (2020). DOI: 10.17504/protocols.io.xy4fpyw; manuscript Nucleic Acids Research 48(19), e112 (2020). DOI: 10.1093/nar/gkaa792. ↩

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