Computational protocol: Comparative Genomic, MicroRNA, and Tissue Analyses Reveal Subtle Differences between Non-Diabetic and Diabetic Foot Skin

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Protocol publication

[…] The formalin fixed, paraffin embedded tissue was cut in 7 μm sections using a microtome. Slides containing sections were deparaffinized with xylene (EMD, Gibbstown, NJ, USA), rehydrated, and hemotoxylin and eosin stained or further processed for immuno-staining as described previously []. Endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol and washed with distilled water. The slides were then incubated in Dako target retrieval solution (Dako, Carpinteria, CA, USA) for 30 minutes at 95°C for antigen retrieval, allowed to cool down, and then treated with Background punisher (MACH1 kit, Biocare Medical, Concord, CA, USA). Primary antibodies were diluted in 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in TBS and applied to the samples (LEPR, Abcam cat # ab5593, 1;1000, CD31, Adb Serotec cat # MCA1746GA, 1:25; CD45, Dako cat # M0701, 1:100; Ki67, Abcam cat # ab15580, 1:500; S100A9 Abcam cat # 22506, 1:100). All antibodies were incubated overnight at 4°C, except for the anti CD45, which was incubated for 30 min at room temperature. The detection and chromogenic reaction was carried out using the MACH 1 Universal HRP-Polymer Detection system (Biocare Medical, Concord, CA, USA) and following manufacturer’s instructions. Picrosirius red (Electron Microscopy Sciences, Hatfield PA, USA) staining was carried out following manufacturer’s instructions. All slides were analyzed with a Nikon Eclipse E 400 microscope and digital images were obtained using a Qimaging camera and NIS-Elements BR3.10 software.Quantification of CD45 positive cells, Ki67 positive cells, and CD31 positive blood vessels was performed using ImageJ software (NIH, Bethesda, NJ, USA). Briefly five (20X) images of each section were taken randomly and the positive cells or structures were counted within a rectangular dermal area of 0.046 mm2 (1000x500 pixels). For Ki67 quantification, positive cells were counted from the total epidermal section and normalized to the epidermal area. […]

Pipeline specifications

Software tools NIS-Elements BR, ImageJ
Applications cryo-EM, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Diabetes Mellitus, Vascular Diseases, Diabetic Foot