Wang et al. BMC Cancer 2010, 10:415 http://www.biomedcentral.com/1471-2407/10/415 USA), 1 ug RNA was used as template for cDNA Synth- esis, cDNA was synthesized with First-Strand Synthesis System for RT-PCR Kit (Catalog No. 11904-018 Invitro- gen). RT-PCR analyses were performed using the follow- ing primer sets, human TLR9(sense: 5′-TGTAATA ACAGTTGCCGTCCAT-3′, antisense: 5′-CAGCCTT TCCTTGTCCT CC-3′, product size:344 bp), rat TLR9 (sense: 5′-GGACAGTTCTCTCCACTCGC-3′, antisense: 5′-TTCTTTGAGACGGGAGTGCT-3′, product size: 509 bp), GAPDH(sense: 5′-TCCACCAC CCTGTTGCTGTA- 3′, antisense: 5′-ACCACAGTCCATGCCATCAC-3′, pro- duct size:450 bp). GAPDH was used as housekeeping gene control, GADPH consensus primers were shared for Homo sapiens and Rattus norvegicus. Reactions were car- ried out in a Gradient Thermal Cycler (Biometra, Goet- tingen, Germany) for 30 cycles which consisted of 94.0°C 30 s, 55°C 30 s, 72°C 30 s. The reaction took place in a total volume of 25 ul. Products were analyzed by 1.5% agarose gel electrophoresis and visualized by Gene finder staining under ultraviolet. All primers were synthezed by Takara Biotechnology (Dalian, China). The expression of TLR9 in U87 and U251 cells were further confirmed by immunoflurescence analysis, and the expression of TLR9 in glioma tissues were further confirmed by immunohis- tochemistry method. Selection of Diffuse Glioma Samples and Construction of Glioma Tissue Microarray The formalin-fixed, paraffin embedded archival tissue blocks were retrieved, and the matching HE-stained slides were screened for representative tumor regions by a neu- ropathologist. Glioma samples were then grouped accord- ing to the diagnostic criteria of the WHO 2000 classification system. The composition of tumors included in the tissue microarray is listed in Table 1. tissue microar- ray was constructed with tissue microarrayer (Beecher Instruments, Silver Springs, MD, USA) as described pre- viously [18]. The tissue microarray included 292 primary gliomas representing different histological types and grades of glioma. Each tumor was sampled in duplicate from representative areas using a 0.6-mm punch, yielding composite array blocks comprising a total of 584 tissue Table 1 Composition of tumor types in glioma tissue array gliomas Glioblastoma (GBM, WHO Grade IV) Anaplastic astrocytoma (AA, WHO Grade III) Diffuse astrocytoma (LGA, WHO Grade II) Anaplastic oligodendroglioma (AO, WHO Grade III) Oligodendroglioma (ODG, WHO Grade II) Anaplastic mixed oligoastrocytoma (AMOA, WHO Grade III) Oligoastrocytoma (MOA, WHO Grade II) non neoplastic brain tissue(NB) Page 3 of 11 cores. In addition, thirteen non-neoplastic brain tissues from epilepsy surgical resections were also included. Nor- mal kidney samples provided by department of surgery were used as positive control. Immunochemistry Paraffin-embedded specimens were cutted into 4 um sections and baked at 65°C for 30 min. The sections were deparaffinized with xylenes and rehydrated. Sections were submerged into EDTA (PH = 8.0) and autoclaved for antigen retrieval, then treated with 3% hydrogen peroxide, followed by incubation with 1% FBS. Anti-TLR9 (Img-305A, clone 26C593.2, Imgenex, San Diego, USA, 1:100 dilutions) was added and incubated overnight at 4°C. For negative controls, the primary antibody was replaced by normal mouse serum. Human normal kidney samples were used as a positive control. Horseradish peroxidase (HRP) labeled secondary anti- body in the MaxVision™ HRP-Polymer antimouse/rabbit IHC kit (KIT-5930 Maixin Biol, Fu Zhou, China) was applied and incubated for 30 mins at room temperature, followed by 5 mins incubation at room temperature with DAB provided in the kit for color development. The sections were finally counterstained with haematox- ylin and mounted with Permount (BIOS, Beijing, China). Results were visualized and photographed under a light microscope (Olympus BX-51; Olympus Optical). The degree of immunostaining of sections was viewed and scored separately by two independent investigators, the scores were determined by combining the propor- tion of positively stained tumor cells and the intensity of staining. Scores from the two investigators were aver- aged for further comparative evaluation of the TLR9 expression. The proportion of positively stained tumor cells was graded as follows: 0 (no positive tumor cells), 1(<10% positive tumor cells), 2 (10-50% positive tumor cells) and 3 (>50% positive tumor cells). The intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light yellow), 2(moderate staining, yel- lowish brown) and 3 (strong staining, brown). The stain- ing index was calculated as follows: staining index = staining intensity × proportion of positively stained Number of cases (tissue cores) 128 (256) 27 (54) 33 (66) 16 (32) 26 (52) 30 (60) 32 (64) 13 (26)