Brief Report H2O2 in citrate buffer, and thoroughly washed in 50 mm Tris-Cl (pH 7.4), 150 mm NaCl Tris buffered saline (TBS) containing 0.05% Tween 20 (washing buffer). Slides were then pre- incubated with 3% bovine serum albumin (BSA) in TBS for 30 min, incubated with 1:300 dilution of anti-PARP-1 antibody in TBS con- taining 1% BSA, thoroughly washed in wash- ing buffer before detection with the LSAB 2 kit (anti mouse, biotinylated and peroxidase- labeled streptavidin) and 3,3-diaminobenzi- dine-4HCl (DAB; Dako, Carpinteria, CA, USA) from Dako, following the instructions con- tained in the kit. After detection, the sections were counterstained with haematoxylin, dehy- drated and mounted in xylen-based DPX moun- tant (BDH, Pool, UK).12 Microscopic evaluation Slides were observed and cells visually scored at 20X and 40X. To evaluate the percent- age of positive tumor cells, microscopic fields were chosen such that non-tumor cells were also included. The fraction of PARP-1 positive cells was evaluated independently in a blinded fashion by two co-authors. No significant dif- ference was observed between the two observers. Statistical analysis Results are expressed as mean±SEM. Data were analyzed by chi-square test and statistical analysis was performed with Graph Pad Prism 5 software (Graph Pad Software, Inc. La Jolla, CA, USA). The statistical significance was accepted for the p value lower than 0.05. Results Normal tissues (4 autopsies and 27 non- tumoral tissue of the same subjects with brain GBM) did not show any nuclear or cytoplasmic signal for the protein PARP-1 (Table 1, Figure 1A). The expression of PARP-1 was also evalu- ated in 27 samples of GBM, PARP-1 positive nuclear signal was present in all 27 samples with the mean of nuclei involved of 87.03±5.04% (P<0.005). No cytoplasmic stain- ing was observed in the samples (Table 1, Figure 1B). The signal was present on the nuclear membrane and nuclear chromatin. Discussion This study showed that PARP-1 protein is detectable in GBM cells, but is undetectable in normal brain tissue. The protein seems to be exclusively localized in the nucleus. This observation suggests that PARP-1 may play a role in this tumor and that the presence of the Table 1. Expression PARP-1 protein in WHO glioblastoma multiforme grade IV astrocytoma. Age (years) Brain localization Histological diagnosis Positive cell nuclei (%) [page 46] [European Journal of Histochemistry 2012; 56:e9] Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Case 11 Case 12 Case 13 Case 14 Case 15 Case 16 Case 17 Case 18 Case 19 Case 20 Case 21 Case 22 Case 23 Case 24 Case 25 Case 26 Case 27 Autopsy 1 Autopsy 2 Autopsy 3 Autopsy 4 Sex Male Male Male Male Male Male Male Male Male Male Male Male Male Male Female Female Female Female Female Female Female Female Female Female Female Female Female Male Male Female Female 41 61 51 67 66 68 60 68 66 53 51 60 38 66 50 69 66 43 62 60 67 28 50 75 62 60 66 66 54 41 70 Right parietal lobe Right temporal lobe Left temporal lobe Right temporal lobe Right frontal lobe Left parietal lobe Left frontal lobe Right frontal lobe Right frontal lobe Right parietal lobe Left temporal lobe Left frontal lobe Left occipital lobe Left occipital lobe Left frontal lobe Left frontal lobe Left frontal lobe Right temporal lobe Right temporal lobe Right frontal lobe Left frontal lobe Right temporal lobe Brain ventricle Right frontal lobe Left frontal lobe Left frontal lobe Left frontal lobe Right frontal lobe Brain ventricle Right parietal lobe Right temporal lobe Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma IV WHO Glioblastoma I VWHO Normal Normal Normal Normal 95 85 90 90 80 85 90 85 90 95 80 85 85 80 95 90 85 90 80 90 95 80 85 90 80 90 85 Not applicable Not applicable Not applicable Not applicable