Sample Pre-treatment: Fresh Frozen¶
- Applicable samples: Fresh Frozen (FF) cryosections
- Version: v6.1
- Last modified: 2026-08-18
What's next?
After completing pre-treatment, proceed to the unified RCA Protocol (Hybridization → Ligation → RCA → Post-fix).
1. Sample Preparation & Sectioning¶
Goal: Obtain high-quality sections free of ice crystals and cracks, with firm adhesion to slides.
- Embedding & Freezing: Use isopentane + dry ice for rapid freezing.
- Cryosectioning: Blade: -20°C, Head: -10°C, Thickness: 10 µm.
- Section Placement: Pre-chilled slide pickup -> finger-back press for adhesion -> return to cold stage immediately.
- Storage: Store at -80°C.
2. Fixation¶
Goal: Brief thermal retrieval for adhesion, then chemical fixation.
- Retrieve: Transport slides on dry ice.
- Thaw & Adhere: Place immediately on 37°C hot plate for 1 min.
- Fixation: Immerse vertically in Slide Mailer with 4% PFA (Fresh), incubate at room temperature (RT) for 30 min.
- Wash: PBS wash 2 x 2 min.
Assemble Chamber: Assemble pre-treatment chamber to limit liquid mount.
3. Permeabilization¶
Goal: Open the fixed protein mesh enough for probes and enzymes to reach the RNA — while leaving the RNA anchored where it is.
- Incubate: 0.1 M HCl at room temperature for 5 min. No protease.
- Wash: PBS-Tween 0.05% wash 2 x 2 min.
New in v6.0, and not yet confirmed at our bench
The pepsin step was removed in favour of acid-only permeabilization. This is the published condition for the two direct-RNA padlock/RCA methods closest to ours, but we have not yet run it side by side against the old one. Run a control section, or the digestion ladder below, before committing a precious sample.
Previous condition, for reference: 0.4 mg/mL pepsin in 0.1 M HCl, 37 °C, 2 min.
Why acid-only — and why the protease was the suspect
An in situ readout only needs probes to reach RNA that stays inside the cell. A capture-based method like Visium needs the RNA to diffuse out to the slide, which is where the original 0.1% pepsin / 10–30 min recipes come from. Conditions inherited from that lineage are systematically too aggressive for padlock + RCA: past the point where RNA is still anchored, the molecule is simply lost during the ~16 h hybridization, the formamide stringency washes and the overnight RCA.
Four independent padlock/RCA systems permeabilize fresh-frozen tissue with no protease at all — SCRINSHOT (0.1 M HCl, 3 min; the same direct-RNA SplintR chemistry as ours), HybISS (0.1 M HCl, 5 min), the commercial CARTANA ISS kit, and Xenium FF (1% SDS + cold 70% methanol). The acid window is also wide: routine FISH slide pretreatment uses 0.2 N HCl for 20 min at room temperature.
Full evidence, citations and the exposure arithmetic are in the lab-private note the internal evidence review Permeabilization / Pepsin Digestion (lab site → Research).
If signal is low: titrate, don't just add pepsin back
Run the one-slide ladder in Permeabilization Optimization (Pilot) — 4–6 serial sections, one PDMS chamber each, every downstream step shared. That page also carries the in-process morphology criteria for judging digestion at the microscope, and the read-out metrics and decision rule for picking a winner.
4. Dehydration¶
Goal: Improve tissue permeability.
- Pre-dehydrate: 80% Ethanol for 10 min.
- Dehydrate: 100% Ethanol for 2 min.
- Rehydrate: PBS-Tween 0.05% wash 2 x 2 min.
Next step: RCA Protocol (Hybridization → RCA)
Changelog¶
-
v6.1 -- 2026-08-18 -- Follows RCA Protocol v6.0, which deleted the blocking step: this page now hands off to hybridization rather than to blocking. MINOR — nothing performed on this page changed, but the stated next step did, and a reader following the old pointer would look for a step that no longer exists.
-
v6.0 -- 2026-08-18 -- Pepsin removed; permeabilization is now 0.1 M HCl for 5 min at room temperature. MAJOR because data produced with and without a protease step are not directly comparable — detection efficiency, and which compartments survive, both change.
Why. A TNBC fresh-frozen run digested at 0.05% instead of the intended 0.04% produced markedly fewer rolonies, which prompted a review of where the step came from. Two findings drove this change. First, an in situ readout only requires that probes reach RNA that remains inside the cell, whereas the capture-based lineage our pepsin recipe descends from requires RNA to diffuse out of the tissue — so those conditions are systematically too aggressive here, and past the point where RNA is still anchored the molecule is lost across the ~16 h hybridization, the formamide washes and the overnight RCA. Second, four independent padlock/RCA systems permeabilize fresh-frozen tissue with no protease at all: SCRINSHOT (0.1 M HCl 3 min, the same direct-RNA SplintR chemistry as ours), HybISS (0.1 M HCl 5 min), the commercial CARTANA ISS kit, and Xenium FF (1% SDS + cold 70% methanol). 5 min was chosen over 3 as the safer starting point for dense tumour stroma: removing the protease moves the risk from over- to under-permeabilization.
Status. Adopted from the literature, not yet validated at our bench — the page carries a warning and a titration ladder to settle it. The v5.3 condition is recorded inline so it can be reinstated or run as a control arm.
Also. The acid is now stated as 0.1 M and the temperature as room
temperature, both explicitly. reagents/pepsin.md previously specified 0.01 M
HCl while every protocol page said 0.1 M; that conflict is resolved in favour of
0.1 M, which is what the entire padlock/RCA literature uses. Neither source paper
states a temperature for the acid step, so RT is stated here rather than left to
be inferred.
Evidence, citations and exposure arithmetic: lab-private note
the internal evidence review Permeabilization / Pepsin Digestion (lab site → Research).
- v5.3 -- 2026-01-29 -- Fresh-frozen pre-treatment split out of the former combined
rca-ff protocol so that sectioning, fixation, permeabilization and dehydration are
documented once and feed the sample-agnostic RCA Protocol. Steps are unchanged
from the combined v5.3; earlier history is in git.