Extracellular DNA Staining: A Method to Identify Extracellular DNA in FFPE Tissue Sections
Transkript
For extracellular DNA staining, first, heat the samples of interest in the slide rack in 60 degrees Celsius for 60 minutes before immersing the slides in two 40 minute changes of solvent in a fume hood. After the second immersion, rehydrate the samples two times in 100% ethanol and one time in 70% ethanol for 5 minutes per treatment.
After the last treatment, use tongs to place the slides into boiling antigen retrieval solution in a pressure cooker on high for 10 minutes. After unmasking the antigen epitopes, transfer the pressure cooker from heat into a sink and immediately run cold tap water over the lid.
Allow the slides to equilibrate for 20 minutes in the antigen retrieval solution before washing the samples two times in 0.01 molar PBS on an orbital shaker for 5 minutes per wash. After the second wash, use a hydrophobic pen to draw circles around the kidney tissue samples and block any non-specific staining with 60 microliters of 10% chicken serum in 5% bovine serum albumin for 30 minutes at room temperature.
At the end of the incubation, carefully replace the blocking solution with 60 microliters of the primary antibody of interest in a humidity chamber overnight at 4 degrees Celsius. The next morning, wash the slides on an orbital shaker in PBS for 2 minutes per wash before adding 60 microliters of an appropriate fluorophore-conjugated secondary antibody to each slide for a 40-minute incubation at room temperature.
At the end of the incubation, wash the slides in PBS as demonstrated, followed by treatment in 0.3% Sudan Black in 70% ethanol to quench any potential formalin-induced autofluorescence. After 30 minutes, wash the slides in tap water to remove any precipitate and immerse the slides in PBS for 10 minutes to prevent any further Sudan Black precipitate formation.
At the end of the incubation, mount the slides onto confocal glass coverslips with 360-microliter drops of mounting solution supplemented with DAPI and seal the coverslips with nail polish. Allow the slides to cure for 24 hours at room temperature. Store them at 4 degrees Celsius protected from light until imaging by confocal laser scanning microscopy according to standard protocols.
