Novolink polymer detection system

Human osteosarcoma tissue sections were deparaffinized with xylene and rehydrated through addition of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval was carried out for all sections in 0.01 M sodium citrate buffer, pH 6 at 95°C for 25 min. Human CTGF, MMP-2, and MMP-3 antibodies were applied at a dilution of 1:200 and incubated at 4°C overnight. The antibody-binding signal was detected using the NovoLink Polymer Detection System (Leica Microsystems) and visualized with the diaminobenzidine reaction. The sections were counterstained with hematoxylin. The immunohistochemistry results were scored by taking into account the percentage of positive detection and intensity of the staining.

The human chondrosarcoma tissue array was purchased from Biomax (Rockville, MD, USA; 6 cases for normal cartilage, 24 cases for grade I chondrosarcoma, 9 cases for grade II chondrosarcoma, and 15 cases for grade III chondrosarcoma). Fixed and paraffin-embedded tissues were deparaffinized with xylene, and rehydrated through a graded series of alcohols to water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide Heat-induced antigen retrieval was carried out for all sections in 0.01 M sodium citrate buffer, pH 6 at 95°C for 20 min. Human AR, integrin α6, and integrin β1 antibodies were applied at a dilution of 1:200 and incubated at 4°C overnight. Bound antibodies were detected by NovoLink Polymer Detection System (Leica Microsystems, Newcastle, UK) and visualized with the diaminobenzidine reaction. The sections were counterstained with hematoxylin. IHC results were scored by taking into account the percentage of positive detection and intensity of the staining.

A prostate adenocarcinoma tissue microarray was obtained from Super Bio Chips (CA4, Seoul, Korea), which contained 49 tissue cores representing samples from 40 cases of prostate cancer and nine matched normal adjacent tissues. After deparaffinization, rehydration, and antigen retrieval, the tissue sections were processed using the Novolink Polymer Detection System (Leica Microsystems, Newcastle Upon Tyne, UK) as previously described^{62 (link), 63 (link)}. Rabbit polyclonal antihuman MMP-3 (1:250 dilution, LifeSpan BioSciences, Seattle, WA) and rabbit polyclonal antihuman MMP-10 (1:400 dilution, LifeSpan BioSciences) antibodies were used for staining. Stained slides were scanned, and the images were digitized using the TissueFAXS system (TissueGnostics, Vienna, Austria) equipped with a 20× objective.

A prostate adenocarcinoma tissue microarray was obtained from IMGENEX (IMT-01254, San Diego, CA) and contained 95 tissue cores representing samples from 48 cases of prostate cancer and 15 matched normal adjacent tissues. Mice bone xenograft tissues were collected at the end of the animal experiments (5 weeks after initial treatment). IHC staining was performed using the Novolink Polymer Detection System (Leica Microsystems, Newcastle Upon Tyne, UK) as previously described [16 (link)]. Antibodies used in the study included mouse anti-human L1CAM (1:40; clone UJ127, NeoMarker), mouse anti-human ki-67 (1:50; NCL-Ki67-MM1, Leica Biosystems), mouse anti-MMP-2 (1:50; clone MMP2/8B4, LifeSpan Biosciences), rabbit anti-MMP-9 (1:100; AB19016, Millipore), and rabbit anti-human NF-κb (1:100; clone E379, Millipore). Scoring of immunostaining of L1CAM in tissue microarray was done by two pathologists from the Department of Pathology, Emory University School of Medicine (Atlanta, GA) using a semi-quantitative scoring method according to the staining intensity and area extent, which has been widely accepted and used in our previous studies [63 (link), 64 (link)]. L1CAM expression was identified as any identifiable cytoplasmic/membranous staining, and quantified in 25% increments by visual estimation. Intensity of staining was recorded as weak, moderate and strong.

IHC staining was performed using the Novolink Polymer Detection System (Leica Microsystems, Newcastle Upon Tyne, UK) as previously described [28 (link)]. Ki-67 protein was detected in tumor xenografts with mouse antihuman Ki-67 monoclonal antibody (1:100; NCL-Ki67-MM1, Leica Biosystems). Apoptosis was evaluated using the Apo-BrdU-IHC In Situ DNA Fragmentation Assay Kit (BioVision, Inc., Milpitas, CA, USA) as described [28 (link)]. IHC staining for osteonectin was performed on prostate TMA using an anti–human-osteonectin monoclonal antibody (1:50; NCL-O-NECTIN, Leica Biosystems). Each TMA spot was examined by a pathologist (J.L.C.) using Allred scoring system [29 (link)]. The numerical value for overall intensity (intensity score) is based on a 4-point system: 0, 1, 2, and 3 (for none, light, medium, or dark staining). The numerical value for percent stained (proportion score) is determined by a geometric division; no stain = 0; ≤1/100 cells stained = 1; ≤1/10 cells stained = 2; ≤1/3 cells stained = 3, ≤2/3 cells stained = 4; all cells stained = 5. Addition of the two values gives the total Allred score [30 (link)].

The tissue slides of osteosarcoma were baked at 60 °C overnight and then deparaffinized using xylene. They were subsequently rehydrated through an ethanol series. To block endogenous peroxidase activity, 3% hydrogen peroxide in methanol was applied for 10 min. Heat-induced antigen retrieval was performed for all sections using 0.01 M sodium citrate buffer at pH 6, heated to 95 °C for 25 min. The rabbit monoclonal antibody for CCL2 (Abcam, Cambridge, MA, USA) and the mouse monoclonal antibody for MMP-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted 1:200 and left to incubate overnight at 4 °C. The antibody-binding signal was detected using the NovoLink Polymer Detection System (Leica Microsystems, Wetzlar, Germany) and visualized using the diaminobenzidine reaction. The sections were counterstained with hematoxylin. The immunostaining intensity of CCL2 and MMP-3 was scored using the MacBiophotonics Image J software (version 1.53) [27 (link)].

Tumor cell sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked in methanol for 10 min with 3% hydrogen peroxide. Antigen retrieval was carried out for all sections in pH 6 sodium citrate buffer 0.01 M at 95°C for 25 min. Human LYVE-1, CCL4 or VEGF-C antibody was applied at a dilution of 1:200 then incubated 2 h. Antibody binding signals were detected with the NovoLink Polymer Detection System (Leica Microsystems) and visualized with the diaminobenzidine reaction. We used Image J software to obtain IHC data, which were semi-quantitatively scored by calculating intensity of the staining and the percentage of positive detection, whereby the results were very low (score of 1: 0–25% of positive area), or weakly (score of 2: 25–50%), moderately (score of 3: 50–75%), or strongly positive (score of 4: 75–100%).