Materials and reagents

Biological materials

1. Human breast cancer samples of surgical specimens of the primary tumor site within the breast, preserved as FFPE tissue blocks, obtained from the Pathology Department of Maastricht University Medical Center+

2. Human lymph node samples of surgical specimens from breast cancer patients undergoing axillary surgery, preserved as FFPE tissue blocks, obtained from the Pathology Department of Maastricht University Medical Center+

Note: After the clearing process, tissue samples are stored in ethyl cinnamate in Falcon tubes at room temperature (RT) until imaging or in the cold room at 4 °C for longer storage.

Reagents

1. Liquid paraffin (histological grade) (Carl Roth, catalog number: 9190.1)

2. Xylene (Laboratorium Discounter, catalog number: XY834.1)

3. Ethanol, absolute (100%) (Carl Roth, catalog number: t913.2)

4. Methanol, absolute (100%) (Carl Roth, catalog number: 4627.4)

5. Milli-Q water

6. Phosphate-buffered saline (PBS), 0.1 M (Carl Roth, catalog number: 9143.2)

7. Thymol (Carl Roth, catalog number: 5391.1)

8. Triton^TM X-100 (octylphenol ethoxylate) (Sigma-Aldrich, catalog number: X100)

9. 30% hydrogen peroxide (H₂O₂) (Carl Roth, catalog number: cp26.1)

10. Potassium disulfite (K₂S₂O₅) (Carl Roth, catalog number: 7995.1)

11. Disodium hydrogen phosphate dihydrate (Na₂HPO₄·2H₂O) (Carl Roth, catalog number: 27ke.1)

12. Citric acid monohydrate (C₆H₈O₇·H₂O) (Carl Roth, catalog number: 3958.2)

13. Neutral Red (NR) (Carl Roth, catalog number: T122.3)

14. Methyl Green (MG) (Sigma-Aldrich, catalog number: M8884)

15. Eosin Y (Morphisto, catalog number: 12199.00100)

16. 4′,6-Diamidino-2-phenylindole (DAPI) stock (1 mg/mL) (Carl Roth, catalog number: 6843.3)

17. Dichloromethane (DCM) (Carl Roth, catalog number: 8424.2)

18. Ethyl cinnamate (ECi) (Sigma-Aldrich, catalog number: 112372)

19. Chloroform (trichloromethane) (Carl Roth, catalog number: 6340.1)

Solutions

1. 50% (v/v) ethanol (see Recipes)

2. 70% (v/v) ethanol (see Recipes)

3. 20% (v/v) methanol (see Recipes)

4. 40% (v/v) methanol (see Recipes)

5. 50% (v/v) methanol (see Recipes)

6. 60% (v/v) methanol (see Recipes)

7. 80% (v/v) methanol (see Recipes)

8. 5% (v/v) hydrogen peroxide (H₂O₂) (see Recipes)

9. 50% (w/w) potassium disulfite solution (see Recipes)

10. McIlvaine buffer (phosphate-citrate buffer), pH 4.0 (see Recipes)

11. NR stock solution, 0.1% (w/v) (see Recipes)

12. MG stock solution, 4% (w/v) (see Recipes)

13. DAPI stock solution, 1 mg/mL (v/v) (see Recipes)

14. Dichloromethane:methanol (DCM) (2:1, v/v) (see Recipes)

15. 0.1 M PBS + 0.2% (v/v) Triton X-100 (see Recipes)

Recipes

See General note 1.

1. 50% (v/v) ethanol

First, measure absolute ethanol (100%) using a graduated cylinder and dilute with Milli-Q water to the final volume. Then, mix thoroughly by gentle agitation. Prepare at the laboratory bench, away from ignition sources, due to ethanol flammability. Store absolute ethanol and all ethanol dilutions at RT in tightly closed, clearly labeled containers in a flammable-liquids storage cabinet, according to institutional safety regulations. Keep containers away from heat sources and open flames.

2. 70% (v/v) ethanol

3. 20% (v/v) methanol

First, measure absolute methanol (100%) using a graduated cylinder and dilute with Milli-Q water to the final volume. Then, mix thoroughly by gentle agitation. Prepare in a chemical fume hood due to methanol toxicity and volatility. Store absolute methanol and all methanol dilutions at RT in tightly closed, clearly labeled containers in a flammable-liquids storage cabinet, according to institutional safety regulations. Due to methanol toxicity and volatility, ensure containers are clearly labeled and minimize opening time during use.

4. 40% (v/v) methanol

5. 50% (v/v) methanol

6. 60% (v/v) methanol

7. 80% (v/v) methanol

8. 5% (v/v) H₂O₂

To prepare a 5% (v/v) hydrogen peroxide solution, measure the required volume of 30% stock using a volumetric pipette and dilute in pre-cooled methanol. Add hydrogen peroxide slowly to the methanol while gently mixing by agitation. Prepare the solution in a chemical fume hood due to the strong oxidative properties of hydrogen peroxide and the potential release of oxygen gas. Protect the solution from light at all times. Prepare the diluted hydrogen peroxide solution immediately prior to use. It is not advised to store diluted solutions, as hydrogen peroxide rapidly decomposes upon dilution and light exposure. Store 30% (v/v) hydrogen peroxide stock at 4 °C in its original, vented, light-protected container, according to the manufacturer’s instructions.

9. 50% (w/v) potassium disulfite solution

First, weigh K₂S₂O₅ using an analytical balance and then add Milli-Q water until just before the final volume. Stir with a magnetic stir while heating the solution up to approximately 60–70 °C. When dissolved, add the remaining Milli-Q to the final volume. Please note that the solution is oversaturated and the salt will precipitate again over time. Avoid inhalation of dust. Store at RT in a tightly sealed glass container. Directly before use, it is advised to stir the solution and heat it up on a magnet stirrer to dissolve the precipitate. This is followed by filtering the clear dissolved solution.

10. McIlvaine buffer (phosphate-citrate buffer), pH 4.0

Prepare 0.2 M Na₂HPO₄ and 0.1 M citric acid stock solutions separately by weighing salts on an analytical balance and dissolving them in Milli-Q water to the required volumes by mixing on a magnetic stir. Combine the two solutions in the specified ratio to achieve pH 4.0 and mix thoroughly. Prepare at the laboratory bench. Store the buffer at 4 °C.

See General note 2.

Step 1. 0.2 M Na₂HPO₄ stock solution

Step 2. 0.1 M C₆H₈O₇·H₂O stock solution

Step 3. Final phosphate-citrate buffer

11. NR stock solution, 0.1% (w/v)

First, prepare a 0.1% (0.1 mg/mL) stock solution by weighing NR using an analytical balance and dissolving it in McIlvaine buffer (pH 4.0). Mix gently until fully dissolved. Dilute the stock dilution 1:100 in McIlvaine buffer (pH 4.0) to obtain the final 0.001% working concentration. Prepare at the laboratory bench under reduced light conditions. Protect from light and store at 4 °C. Store NR powder at RT in a tightly closed container, protected from light and moisture. See General note 3.

12. MG stock solution, 4% (w/v)

First, prepare a highly concentrated 4% MG aqueous stock solution according to [13]. Remove crystal violet impurities by extractions with chloroform in a separation funnel, thereby discarding the lower (violet) phase until the lower chloroform phase no longer contains the violet color. Dilute the stock solution 1:5,000 in McIlvaine buffer (pH 4.0). Prepare at the laboratory bench under reduced light conditions. Protect from light and store at 4 °C. Store MG powder at RT in a tightly sealed container, protected from light and humidity. See General note 3.

13. DAPI stock solution, 1 mg/mL (v/v)

First, prepare a 1 mg/mL stock solution by weighing DAPI using an analytical balance and dissolving it in McIlvaine buffer (pH 4.0). Mix gently until fully dissolved and minimize light exposure. Store DAPI stock solutions at -20 °C, protected from light, according to the manufacturer’s instructions. Dilute the DAPI stock solution 1:1,000 in McIlvaine buffer (pH 4.0) to obtain the final 1 μg/mL working solution. Prepare at the laboratory bench immediately prior to use. See General note 3.

14. DCM (2:1, v/v)

Measure DCM and methanol using glass graduated cylinders and combine in a 2:1 (v/v) ratio. It is advised to prepare this strictly in a chemical fume hood due to the high volatility and toxicity of both dichloromethane and methanol. Stir until the inhomogeneities disappear. Store dichloromethane in tightly closed, chemically compatible containers at RT in a dedicated halogenated-solvent storage cabinet, separate from flammable solvents. Ensure containers are clearly labeled and kept away from heat sources. See General notes 4 and 5.

15. 0.1 M PBS + 0.2% (v/v) Triton X-100

Prepare 0.1 M PBS using MilliQ water. Triton X-100 is highly viscous, which can complicate pipetting. To facilitate handling, the end of the pipette tip may be cut. Solutions containing Triton X-100 should not be shaken; instead, gently stir and allow the detergent to dissolve passively, which may take up to 1 h.

Laboratory supplies

1. Glass containers, 100, 250, and 500 mL

2. Glass Petri dish

3. Beakers, 50 and 200 mL

4. Erlenmeyer flask, 50 mL

5. Graduated cylinders, 25, 50, 100, 250, and 500 mL

6. 50 mL Falcon tubes

7. Six-well cell culture plates

8. Pipettes, 0.2, 1, and 5 mL, with compatible pipette tips

9. Forceps

10. Filter paper

11. Funnel

12. Millimeter paper

13. Nitrile gloves

Equipment

1. Water bath, set to 60 °C (Gesellschaft für Labortechnik mbH, model: 1002)

2. Fume hood (Vinitex Laboratoriuminrichtingen B.V., model: L1K1-GR+51)

3. Chemical waste disposal system (institutional facility)

4. Orbital shaker (Polymax, model: 1040)

5. Cold room or refrigerated storage, 4 °C

6. Analytical balance (Mettler Toledo, model: PG2002-S DeltaRange)

7. Magnetic stirrer (Framo®-Gerätetechnik, model: M21/1)

8. Led-light pad (optional) (Crafts & Co, Karsten International, catalog number: 973)

9. Two-photon (2P) laser scanning microscope (Leica TCS SP5 MP, Leica Mikrosysteme Vertrieb GmbH)

10. UltraMicroscope II light-sheet microscope (La Vision Biotech, Miltenyi)

Software and datasets

1. Leica Application Suite Advanced Fluorescence (Leica Microsystems), for Leica 2-photon microscopy data

2. Imaris Biplane 11.0, for 2-photon microscopy and LSFM 3D volume data

3. FIJI (open-source Bio-image analysis software)

Procedure