Extracellular DNA Staining: A Method to Identify Extracellular DNA in FFPE Tissue Sections

1. Staining for ecDNA with DAPI and β-Actin

1. Put glass slides into a slide rack in a 60 °C oven for 60 min.
2. Immerse slides in two changes of solvent (xylene or substitute) for 40 min each, in a fume hood.
3. Rehydrate slides in 100% ethanol, 100% ethanol, and 70% ethanol for 5 min each. Wash for 5 min in tap water.
4. Place a hot plate in a fume hood and pre-boil antigen retrieval solution (10 mM Tris, 1 mM EDTA pH 9.0) in a pressure cooker. As soon as the antigen retrieval solution starts to boil, place the slides in the pot horizontally using a pair of tongs and lock the lid. Boil on high (equivalent to 15 psi or 103 kPa) for 10 min.
   NOTE: This step is crucial as it retrieves the antigen of interest by unmasking the antigen epitope (cross-linked via formalin fixation) to allow epitope antibody-specific binding; the subsequent staining will not work without it.
5. Remove pressure cooker from heat and remove lid immediately by running cold tap on top of the lid in a sink. Leave slides to equilibrate for 20 min in the antigen retrieval solution.
6. Wash slides twice for 5 min in 0.01 M phosphate-buffered saline (PBS) (pH 7.4) on an orbital shaker.
7. Use a hydrophobic pen to draw circles around the kidney tissue being careful not to let any tissue dry out in this time.
8. Block tissue in 10% chicken sera in 5% bovine serum albumin (BSA)/PBS (pH 7.4, 0.01 M) for 30 min, 60 µL per section. Do not wash after this step but carefully remove the blocking solution using a 60 µL pipette, do not let the sections dry out.
9. Apply primary antibody cocktail for β-actin diluted 1/1000 in 1% BSA/PBS (pH 7.4, 0.01 M), 60 µL per section and incubate overnight in a humidity chamber at 4 °C.
10. Wash slides twice for 5 min in PBS (pH 7.4, 0.01 M) on an orbital shaker.
11. Apply secondary antibody, chicken anti-rabbit 488 (1/200), 60 µL per section diluted in 1%BSA/PBS (pH 7.4, 0.01 M) for 40 min at RT.
12. Wash the slides twice for 5 min in PBS (pH 7.4, 0.01 M) on an orbital shaker.
13. Immerse the slides in 0.3% Sudan Black in 70% ethanol for 30 min in a Coplin jar.
    NOTE: This step is important as it quenches the autofluorescence caused by formalin fixation.
14. Wash the slides in tap water to remove precipitate and then immerse in PBS (pH 7.4, 0.01 M) for 10 min to prevent further Sudan Black precipitate from forming.
15. Mount slides onto confocal glass coverslips using three 60 µL drops of mounting solution with DAPI and seal the coverslips with nail polish by applying around the perimeter of the coverslip.
16. Allow slides to cure for 24 hours at RT (to allow DAPI to penetrate) and then store at 4 °C protected from light.
    NOTE: Important to keep in the dark to prevent fading of fluorescent probes.
17. Image slides using a confocal laser scanning head attached to a microscope. Excite slides with the 405 laser for DAPI and the 488 Laser for Beta Actin. Capture single plane 1024 x 1024 pixel images by using line-sequential capturing and 4-line averaging, which is essential for detecting ecDNA. 40x 1.0 NA oil objective is used.
    1. Image a minimum of 20 glomeruli per sample. Save images as ND2 files.