Computational protocol: Connective tissue growth factor-specific monoclonal antibody inhibits growth of malignant mesothelioma in an orthotopic mouse model

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Protocol publication

[…] Whole cell lysates and homogenized tissue lysates were prepared in ice-cold Lysis-M buffer (Roche, Mannheim, Germany), supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche). The whole process after protein separation was performed as previously described []. We used 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for protein separation. The primary antibodies and the dilutions used were: CTGF (Santa Cruz Biotechnology, Santa Cruz, CA; 14939, 1:200), X-linked inhibitor of apoptosis (Abcam, Cambridge, UK; 21278, 1:1,000), β-actin (Sigma-Aldrich, St. Louis; A1978, 1:1,000). Secondary antibodies were immunoglobulins conjugated to horseradish peroxidase (Dako, Glostrup, Denmark; 1:1,000). Immunodetection was performed using Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Densitometric analysis was performed using the ImageJ software (http://rsb.info.nih.gov/ij/). [...] Fibrosis was detected by light green in Elastica-Masson staining. All xenograft histological specimens stained by light green were digitized, using the microscope and camera described above. We analyzed the image using the ImageJ software and color deconvolution plugin (http://imagej.net/Colour_Deconvolution) for ImageJ, and Fiji implements staining separation by the method of Ruifrok and Johnston []. Extracted light green-positive area was converted to black (threshold: upper cutoff, 200; lower cutoff, 0) with reduction of noises. After these processes, most of the remaining dots in the image were those originally stained in light green, and we thereafter measured the whole area of the dots. This area was divided by the entire tumor mass area and the percentage of fibrosis was obtained. [...] The data were analyzed using SPSS Statistics 24.0 (IBM, Armonk, NY). Statistical significance between two groups of interest was analyzed using the unpaired Student's t-test if not indicated otherwise. The results were shown as the mean ± SEM except for where noted. A P value of < 0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, Colour deconvolution, SPSS
Applications Miscellaneous, Bright-field microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Mesothelioma, Neoplasms, Pancreatic Neoplasms