Computational protocol: Activation of PPARγ in Myeloid Cells Promotes Lung Cancer Progression and Metastasis

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Protocol publication

[…] For immunofluorescence labeling, PFA-fixed, paraffin-embedded tissues were deparaffinized, rehydrated, and underwent antigen retrieval by heating for 20 min at 115°C in a decloaking chamber (Biocare). Sections were then exposed to specific antibodies overnight at 4°C. After incubations with primary antibodies, antigen/antibody complexes were visualized using Alexa Fluor-568–coupled, Alexa Fluor-488–coupled, or Alexa-647-coupled secondary antibodies (Molecular Probes); for triple immunofluorescence, sections were sequentially incubated with specific primary and secondary antibodies. Coverslips were mounted with VectaShield medium containing 4′,6-diamidino-2-phenylindole to detect all cell nuclei (Vector Laboratories). Sections were visualized using a Nikon inverted fluorescence microscope equipped with Metamorph software or using a laser-scanning confocal microscope (510 META NLO, Carl Zeiss, Thornwood, NY) with LSM 510 software. Negative controls included the use of mouse or rabbit IgG. Antibodies used include polyclonal FITC-conjugated anti-GFP (1∶200; Abcam), monoclonal anti-Mac3 (1∶50; BD Pharmingen), and polyclonal anti-Arginase I (1∶200; Santa Cruz). Quantification of macrophage recruitment was determined by counting the number of Mac3-positive cells in 40× fields surrounding primary tumors or within tumors. Three 40× fields were counted per section; three sections per animal were counted. [...] Bone marrow–derived macrophages (2×106) from either WT, PPARγflox/flox or PPARγ-Macneg mice were isolated and grown on the bottom of 6-well plates for 4 days in the presence of M-CSF as previously described , . CMT/167-luc cells (3×104) were grown on Transwell filters containing 0.4 mm pores (Corning). Cells were placed in co-culture for 3 days, and lysates were prepared in RIPA buffer and immunoblotted for arginase I or iNOS. Arginase I antibody (Santa Cruz) was used at 1∶200, iNOS antibody (BD) was used at 1∶1000 and b-actin (Sigma) was used at 1∶5000 as a loading control. As a positive control macrophages were stimulated for 24 hours with 20 ng/ml interferon-g/100 ng/ml LPS to induce the M1 phenotype, and 20 ng/ml IL-4 (Sigma) to induce the M2 phenotype. Relative abundance of protein was determined by quantitative densitometry using ImageJ software (NIH, Bethesda, MD). All Western Blot densitometry data on arginase I were normalized to b-actin. The relative level of arginase I was then normalized by the level of arginase I in WT bone marrow-derived macrophages co-cultured with CMT/167-luc cells in the absence of pioglitazone. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Carcinoma, Non-Small-Cell Lung, Lung Neoplasms, Neoplasms
Chemicals Thiazolidinediones