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

Overview

This video describes the technique of staining extracellular DNA in FFPE tissue sections. The stained extracellular DNA can be quantitated to determine the efficacy of any therapeutic treatment that leads to the death of cancer cells.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
Bovine Serum Albumin	SIGMA	A2153	5% and 1% solutions are made up in PBS, can be made in bulk and frozen - discard once thawed
Chicken anti Goat IgG (H+L) cross absorbed antibody Alexa Fluor 594	ThermoFisher Scientific	A-21468	Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail
Chicken anti mouse IgG (H+L) cross absorbed antibody, Alex Fluor 647	ThermoFisher Scientific	A-121468	Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail
Chicken anti rabbit IgG (H+L) cross absorbed antibody Alexa Fluor 488	ThermoFisher Scientific	A-21441	Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail
Chicken sera	SIGMA	C5405	Made up in 1% BSA/PBS
Coverslips 24x60 mm	Azerscientific	ES0107222	#1.5 This is not standard thickness designed for use in confocal microscopy
EDTA 10mM	SIGMA	E6758	Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x Solution
Ethanol 30%, 70% and 100%	Chem Supply	UN1170	Supplied as 100% undenatured ethanol - dilute to 30% and 70% using distilled water
Formaldehyde, 4% (10% Neutral buffered Formalin)	TRAJAN	NBF-500	Kidney is put into a 5ml tube containing 3ml of formalin for 16 hours at RT, formalin should be used in a fume hood
Glass histology slides- Ultra Super Frost, Menzel Glaze, 25x75 x1.0mm	TRAJAN	J3800AM4	Using positive charged coated slides is essential. We do not recommend using poly-L-lysine for coating slides as tissue dislodges from slides during the antigen retrieval step
Goat anti human/mouse MPO antibody	R&D	AF3667	Aliquot and freeze at minus 80 degrees upon arrival
Hydrophobic pen	VECTOR Labs	H-400	Use to draw circle around kidney tissue
Phosphate Buffered Saline	SIGMA	P38135	0.01M PB/0.09% NaCl Make up 5L at a time
Pressure Cooker 6L Tefal secure 5 Neo stainless	Tefal	GSA-P2530738	Purchased at local homeware store
Prolong Gold DAPI	Life Technologies	P36962	Apply drops directly to coverslip
Rabbit anti human/mouse Beta Actin antibody	ABCAM	ab8227	Aliquot and freeze at minus 80 degrees upon arrival
Rabbit anti human/mouse H3Cit antibody	ABCAM	ab5103	Aliquot and freeze at minus 80 degrees upon arrival
Staining rack 24 slides	ProScitech	H4465	Staining rack chosen has to be able to withstand boiling under pressure and incubation in 60 degree oven
Sudan Black B	SIGMA	199664	0.3% Add 3g to a 1L bottle in 70% ethanol, filter and protect from the light - stable for 6 months at room temperature
Tris 10mM	SIGMA	T4661	Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x solution
Xylene	Trajan	XL005/20	Must be use used in a fume hood