The accompanying images are representative projections of confocal z-series with the number of apoptotic nuclei closest to the corresponding averages shown in Figure 3A. Apoptosis in the brains of 58 hpf untreated controls was negligible, and heat shock preconditioning alone did not increase this value . However, 100 min of hypoxia in the absence of heat shock preconditioning at 48 hpf increased the number of apoptotic nuclei detected at 58 hpf to an average of 23.8+/22.9 nuclei per unit volume in the eye and 15.3+/21.4 nuclei in the brain. In contrast, the number of apoptotic nuclei per unit volume in heat shock preconditioned embryos was 12.5+/20.6 and 11.0+/20.3 . These latter values were statistically different from, and approximately 70% less than, values SCH772984 obtained from tissues of embryos subject to HR without preconditioning. To confirm that heat shock protein expression in brain and eye tissues were elevated by heat shock preconditioning, we conducted immunolocalization of Hsp70 and Hsp27 in these tissues . For both Hsp70 and Hsp27, Trichostatin A fluorescent signal was increased in eye and brain following heat shock preconditioning. Additionally, similar to results shown in Figure 2B, preconditioning increased the expression of Hsp27 to a greater extent than that of Hsp70. To investigate the role of HSF1 in zebrafish embryos after HR, we conducted loss of function studies by injecting zebrafish embryos with a previously described antisense HSF1 morpholino oligonucleotide designed to specifically inhibit translation of zebrafish HSF1 mRNA . The previous authors showed that injection of zebrafish embryos with this MO prevented gel mobility shift of radiolabeled nucleotides containing a heat shock response element when incubated with embryo lysates. Here, we examined HSF1 expression in embryos by Western blotting. Figure 4A shows a representative Western blot of protein obtained from non-transfected HeLa cells and cells transfected with plasmid coding for expression of an enhanced green fluorescent protein alone or a zebrafish HSF1- EGFP fusion protein. A band is detected in all samples at the predicted molecular weight of human HSF1 , and another is seen at the predicted molecular weight of the zebrafish HSF1-EGFP fusion protein in samples obtained from cells transfected with plasmid coding for the fusion protein, but not for EGFP alone. Figure 4B is a representative Western blot of nuclear protein in 48 hpf embryos following injection with a 0.25 mM solution of either a control or a-HSF1 MO.