Coolsnap es bx51 camera

TUNEL assay was performed on tibial growth plate sections of three-week old mice using a fluorometric TUNEL kit (Promega, G3250) according to manufacturer's protocol as describe previously [49 ].The slides were mounted with Vectashield with DAPI (Vector Laboratories, H1200) and imaged using the CoolSNAP ES Olympus BX51 camera and associated Metaview software. In order to obtain a reliable estimate of number of apoptotic hypertrophic cells in the growth plate, the green fluorescent apoptotic cells within the HZ were counted in three slides, at least 50 μm apart, per mouse and five animals for each genotype. The total number of hypertrophic chondrocytes within the HZ was defined by the number of DAPI-stained nuclei (as determined using ImageJ software). The apoptosis rate was defined as a percentage of the total number of hypertrophic cells within HZ. All data were analysed by one-way ANOVA for statistical significance.

Cells containing fragmented DNA were evaluated in paraffin sections using a fluorometric TUNEL kit (Promega, Madison, WI, USA, G3250) according to the manufacturer's protocol. Briefly, the sections were dewaxed in xylene and rehydrated through a decreasing series of ethanol concentrations in water. The sections were immersed in 0.85% NaCl for 5 min, washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min and washed twice in PBS. To permeabilize the sections and allow antigen unmasking, the slides were treated with hot (>85 °C) citrate buffer for 10 min and then cooled for 20 min before being washed with PBS. The sections were incubated with 100 μl of equilibration buffer for 10 min and then labeled with 50 μl of TUNEL reaction mix for 1 h at 37 °C. The reactions were stopped by a 15-min wash in × 2 saline-sodium citrate buffer. The sections were washed in PBS to remove excess label and then dyed with 5 μg ml⁻¹ Hoechst 33342 (BD Biosciences) for 30 min at room temperature. After washing in PBS, the slides were mounted with Vectashield, and the images were collected using a CoolSNAP ES Olympus BX51 camera with the associated Metaview Software (Tokyo, Japan).