Next, we assayed expression of representative members of the high and low molecular weight HSPs Hsp70 and Hsp27 in embryos subjected to heat shock preconditioning , hypoxia/reperfusion injury , or both. These proteins have been previously implicated in protection of cells against ischemic injury . Because 160 min of hypoxia was lethal to a majority of 48 hpf embryos , embryos in this study were exposed to hypoxic conditions at 48 hpf for only 100 min. This treatment was sublethal for 100% of the embryos . Heat shock protein expression was evaluated at 58 hpf. Heat shock preconditioning resulted in an increase in Hsp70 and Hsp27 expression in embyros . Hypoxia/reperfusion injury also resulted in Nilotinib increased expression of these proteins . Finally, combined heat shock preconditioning and subsequent HR also elevated Hsp70 and Hsp27 expression relative to untreated controls . However, it does not appear that the combination of stresses produced an additional increase in Hsp70 expression, relative to that produced by FTY720 individual stresses. In contrast, treatment of embryos with both stressors leads to higher expression of Hsp27 than individual stressors. Interestingly, our results suggest that Hsp27 expression is increased in zebrafish embryos exposed to heat shock preconditioning to a greater extent than that of Hsp70, while Hsp70 expression appears to be more responsive to HR. The results shown are representative of three independent trials and blots. The above studies demonstrate that zebrafish embryos display a heat shock response that is protective against subsequent lethal HR. However, clinically relevant brain and eye impairment in humans occurs in response to local, sublethal ischemia/ reperfusion injury. To examine the role of the heat shock response in zebrafish embryos exposed to this type of injury, we compared apoptotic cell death in brain and eye tissues of embryos exposed to sublethal hypoxia with and without heat shock preconditioning . Quantitative analysis of the average number of apoptotic nuclei per unit volume is shown in Figure 3A.