Deadend fluorometric tunel assay

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).

Silanized slides containing spleen sections were fixed in 10% paraformaldehyde in PBS for 15 minutes at room temperature. They were then washed twice in PBS for 5 minutes each. Cell permeabilization was performed with proteinase K (Invitrogen, Carlsbad, CA, USA, final concentration of 20 μg/mL) in 10 mM Tris/HCl pH 8.0 reconstituted (10 mg/mL) and diluted 1:500 in PBS. A volume of 100 μl of proteinase K (20 μg/mL) was added to cover the section of tissue for 10 minutes at room temperature. The slides were washed in PBS, and the TUNEL reagent (DeadEnd™ Fluorometric TUNEL assay, Promega Co., USA) was added to the sections according to the manufacturer’s instructions. This reagent allows the detection of DNA fragmentation by labelling the terminal portions of nucleic acids. For the negative control, only the equilibration buffer was added. Incubation in a humid chamber was performed at 40 °C for 90 minutes. The slides were subsequently washed with PBS for 5 minutes under constant stirring, and after drying, they were mounted in Fluoromount-G with DAPI (Thermo Fisher Scientific, USA). The slides were analysed under a Zeiss fluorescence microscope, and the labelled apoptotic cells were quantified to their full extent in alternating fields under 40x magnification. The total number of DAPI-labelled cells was also quantified.