Filters were dehydrated, cleared twice for 1 min each in 95% ethanol, 100% ethanol and then xylene, cut in half, recleared in xylene for another minute, mounted on microscope slides with a counting grid using Permount™ Mounting Medium Fisher Scientificc, Hampton, NH) and dried flat.
Once the agarose polymerized, samples were post-fixed in 4% PFA for an additional 30 minutes, mounted, and vibratome sectioned in the coronal plane at 100 µm.
After 10 minutes, cells were fixed with 4% paraformaldehyde for 10 minutes, mounted onto glass slides with Mowiol mounting medium, and observed under a Zeiss fluorescence microscope by using excitation 510 nm/emission 580 nm.
Cells were counterstained with DAPI for 3 minutes, mounted on glass slides, and observed using a fluorescence microscope (Olympus B×51, Japan) to determine the intensity of the uptake of latex beads by visual examination.
Finally crystals were placed in 40% PEG3350 solution for a few minutes, mounted on loops and frozen in liquid nitrogen.
Sections were rinsed in KPBS (3×, 10 minutes), stained with 0.0002% DAPI for 5 minutes, rinsed again in KPBS (3×, 10 minutes), mounted onto SuperFrost slides (Fisher Scientific), and coverslipped with antifade medium.
The cells were also costained with a 1 : 500 dilution of DAPI in PBS for 2 minutes, mounted using a fluorescence mounting medium (DAKO North America, Inc, Carpinteria, CA, USA), and observed under a fluorescence microscope Leica AF6000.
For γ-H2AX, cells were counterstained with 2 μg/mL propidium iodide for 2 min. Slides were then rinsed in distilled water for 30 minutes, mounted with Vectashield® (Vector Laboratories, Peterborough, UK) and the edges sealed with clear nail polish.
Cells were washed in PBS for 10 minutes, mounted between two glass slides with Fluoromount (Sigma-Aldrich), and imaged using an Olympus FV-1000 with a 60× lens and a 4× digital zoom.
The next day, sections were rinsed in KPBS (3x, 10 minutes), stained with 0.0002% DAPI for 5 minutes, rinsed again in KPBS (3×, 10 minutes), mounted onto SuperFrost slides (Fisher Scientific), and coverslipped with antifade medium composed of 96 mM Tris HCl, pH 8.0, 24% glycerol, 9.6% polyvinyl alcohol, and 2.5% diazabicyclooctane (Sigma-Aldrich).
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