Alexa fluor 594 anti mouse igg

All sections were dried, rehydrated in phosphate buffered saline (PBS), and rinsed in PBS. Sections were blocked for 60 min in 5% BSA (PBS containing 5% BSA and 0.2% Triton X-100; Sigma) and incubated with the appropriate primary antibodies, either mouse monoclonal anti-NeuN antibody (Millpore; 1:500, NeuN is a neuronal marker)/ anti-p53 antibody (GeneTex ; 1:500) / anti-annexin V antibody (Abcam ; 1:500) / anti-p-p53 antibody (Cell signaling ; 1:1000) / or anti-PUMA antibody (Abcam ; 1:200) at 4°C overnight and with secondary antibodies (Alexa Fluor® 488 goat anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA); Alexa Fluor® 594 anti-mouse IgG (1:200 dilution, Jackson ImmunoResearch, West Grove, PA)) at room temperature for 2 h. Sections were mounted with Mounting Medium H-1000 (Vector Laboratories, Burlingame, CA, USA). The numbers of NeuN-, p53-, annexin V-, p-p53-, and PUMA–positive cells were counted in 5 randomly selected fields by means of SPOT image analysis software (Diagnostic Instruments, Sterling Heights, MI)

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.