Rabbit polyclonal anti puma

To evaluate co-localization of neuronspecific nuclear protein (NeuN) with p53, annexin V, or 4-hydroxynonenal (4-HNE) and PUMA with COX IV, we performed double immunofluorescence staining. For immunofluorescence staining, adjacent sections to those stained with HE were then blocked for 60 min in 5% BSA (Sigma Chemical Co., St Louis, MO, USA). Thereafter, the following primary antibodies were used and incubated overnight at 4°C: (i) mouse monoclonal anti-NeuN (Millpore; 1:500), (ii) rabbit polyclonal anti-p53 (GeneTex; 1:500), (iii) rabbit polyclonal anti-annexin V (Abcam; 1:500), (iv) rabbit polyclonal anti-4-HNE (Abcam; 1:250), (v) rabbit polyclonal anti-PUMA (Abcam; 1:1000), and (vi) mouse monoclonal anti-COX IV (Abcam; 1:250) antibody. Following incubation with the primary antibody, the sections were washed and incubated with Alexa Fluor® 488 goat anti-rabbit IgG (Jackson; 1:200) and Alexa Fluor® 594 anti-mouse IgG (Jackson; 1:200) at room temperature for 2 h. Sections were then mounted with Mounting Medium H-1000 (Vector Laboratories). The numbers of NeuN/p53-, annexin V-, 4-HNE and PUMA-COX IV positive cells were counted in three sections by means of SPOT image analysis software (Diagnostic Instruments, Sterling Heights, MI). Control samples were generated by omitting the respective primary antibodies.