Axio hxp 120

Cell death was determined in retinal sections by DeadEnd™ Fluorometric TUNEL assay following the manufacturer’s instructions (Promega, Madison, WI, USA). Nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Invitrogen, Carlsbad, CA, USA) (1:2000). The preparations were mounted with Glycergel mounting medium (DAKO, Agilent, Santa Clara, CA, USA) and were observed in an inverted fluorescence microscope (Zeiss Axio HXP-120, Zeiss, Oberkochen, DE) with a 20x objective (Plan Achromat 20×/0.8 M27). The number of TUNEL-positive cells was counted in the entire retinal section (4 sections per eye).

Microglia migration was assessed in BV‐2 cells and in primary microglia using the Boyden chamber migration assay as described (Siddiqui, Lively, Vincent, & Schlichter, 2012). BV‐2 cells were kept in serum‐free medium for 24 hr before beginning the experiment. Cells (BV‐2 cells in 2% FBS or primary microglia in 10% FBS) were plated in transwell culture inserts with 8.0 μm pore diameter (Merck, Millipore, Billerica, MA) at a density of 3 × 10⁴ cells/cm² and then cultured for 4 hr in control or EHP conditions. Inserts were washed with warm PBS and following fixation in 4% paraformaldehyde with 4% sucrose for 10 min the cells in upper side were removed with a cotton swab. Nuclei were stained with DAPI (1:2,000) to allow cell counting. The membrane was removed and mounted with Glycergel in glass slides. The preparations were observed in an inverted fluorescence microscope (Zeiss Axio HXP‐120, Oberkochen, DE) and five random images per preparation were acquired (10× objective). The number of cells in the bottom side of the insert (the cells that migrated) was counted.