Cdd camera

Immunohistochemical staining was performed on tissue microarray (two cores for one tumor block using primary antibodies against human CCL21 (Anti-CCL21 antibody, ab9851, abcam, diluted 1/500)) and visualization reagent (DakoEnVision Detection System) as previously described [30 (link)]. Antibody specificity was confirmed by immunochemistry and western blot. Olympus CDD camera, Nikon eclipse Ti-s microscope (×400magnification) and NIS-Elements F3.2 software were used to record the staining results. We took three independent shots with the strongest staining for each tumor core. The intensity of immunohistochemical staining of CCL21 was scored by two urologists unaware of the patients’ clinical features and outcomes using Image-Pro Plus version6.0 software (Media Cybernetics Inc., Bethesda, MD, USA). The kappa value was analyzed for evaluating inter-observer agreement. The pooled IOD mean of each patient's 2 cores (6 scans) was regarded as the staining intensity for each block. The IOD score from two observers were averaged again for final statistical analyses.

We constructed tissue microarrays (two cores for one tumor block) with formalin-fixed, paraffin embedded surgical specimens. Immunohistochemical staining was performed on tissue microarrays with protocols described previously [17 (link)]. Antibodies against CCL17 (Anti-TARC antibody, ab182793, Abcam, diluted 1/100) and visualization reagent (DakoEnVision Detection System) were used. The specificity of the antibody was confirmed by western blot using RCC cell lines. We used Olympus CDD camera, Nikon eclipse Ti-s microscope (×200magnification and × 400magnification) and NIS-Elements F3.2 software to record the staining results. We took three independent shots and chose the strongest for each tumor core. The intensity of immunohistochemical staining of CCL17 was scored by two urologists unaware of the patients’ clinical features and outcomes using Image-Pro Plus version6.0 software (Media Cybernetics Inc., Bethesda, MD, USA). The pooled IOD mean of the six spots in two tumor cores was regarded as the final staining intensity for each block. We defined IOD score of 8461 as the cutoff value for high and low expression with X-tile software according to the ‘minimum P-value method’ on the basis of its relation with OS [18 (link)].

Immunohistochemical staining was performed on tissue microarray (TMA) with antibody against Dot1l (ab64077, Abcam, 1:100 dilution) and proper visualization reagent (DakoEnVision Detection System) as previously described [20 (link)]. Olympus CDD camera, Nikon eclipse Ti-s microscope (×200 magnification) and NIS-Elements F3.2 software were used to record the results. The staining intensity and extent was scored by two independent pathologists without the knowledge of the patients’ outcomes. A semiquantitative H-score, ranged from 0 to 300, was used for each sample by evaluating the staining intensities (0: negative, 1: weak, 2: moderate, 3: strong) and distribution areas (0-100%). Three independent shots with strongest staining were selected for each core and the mean score of the three shots was regarded as the final staining intensity for each sample. The H-score cutoff point for determining tumoral Dot1l high/low expression is 95, which was evaluated by X-tile software (Yale University School of Medicine, New Haven, CT, USA) using minimum p value method [21 (link)].