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[…] Harvested tissue was placed into 10% (v/v) neutral buffered formalin (American Master Tech Scientific Inc., Lodi, CA) and incubated at 4°C overnight. Tissue was washed three times with PBS, three times with 70% (v/v) ethanol and stored at 4°C in 70% (v/v) ethanol. All tissue processings were completed using a rapid microwave histoprocessor (Micron Instruments, Inc. Carlsbad, CA). Tissues were embedded in paraffin (Leica Biosystems, Buffalo Grove, IL) and 5-µm sections were placed onto Superfrost Plus slides (Fisher Scientific). Immunohistochemical and H & E staining were performed on deparaffinized and rehydrated sections. Immunohistochemical staining for rabbit anti-human MPO (Dako, Cat #A0398) was carried out as previously described () using heat-mediated antigen retrieval in a Tris-EDTA buffer at pH9.0. For, secondary detection of the primary antibody, we used biotin-conjugated goat anti-rabbit antibody followed by HRP-conjugated streptavidin for 3,3’-diaminobenzidine conversion according to Vector Elite ABC staining kit (Vector, Burlingame, CA). Nuclei were counterstained with Mayer’s hematoxylin for brightfield visualization and coverslips mounted with Cytoseal XYL (ThermoFischer, Waltham, MA).Immunofluorescent staining was performed on flash frozen tissue collected in Tissue-Tek OCT (Sakura, Torrance, CA) and frozen on dry ice. 8 μm sections were collected on a Leica CM1900 cryostat at 20°C. Tissue was subsequently fixed to slides with ice-cold acetone for 15 min and washed in PBS. Slides were incubated with primary antibodies overnight: rat anti-mouse CD86 (1:100, BD Biosciences, Cat #553698), goat anti-mouse CD206 (1:1000, R and D Systems, Cat #AF2535), rabbit anti-human CD3 (1:400, Dako, Cat #A0452), rabbit anti-mouse IBA1 (Wako, Cat #019–19741). Secondary detection of antibodies was carried out using donkey antibodies conjugated to Alexa Fluor 488, 594 (1:500, Invitrogen, Carlsbad, CA) Nuclei were counterstained with 10 µg/ml DAPI and coverslips were mounted using ProLong Gold mounting medium (Invitrogen, Carlsbad, CA) for fluorescence.To quantify the total area of positive signal for fluorescent images, three photomicrographs within the center of the injury were obtained at 40x magnification using an Olympus BX53 fluorescent deconvolution microscope (Olympus America Inc). Quantification of positive signal was performed on four separate samples (one ear per animal) per time point (unless otherwise noted) by thresholding fluorescent signal and mask subsampling with Metamorph Imaging software (Molecular Devices, Sunnyvale, CA). The ratio of total immuno-positive area per total area of the region of interest was then calculated. To quantify total number of DAB-positive cells, two photomicrographs within the center of the injury were obtained at 20x magnification using an Olympus BX53 microscope. Counts were calculated using the Cell Counter plugin for ImageJ (version 1.51d). Cells included in counts were based on both nuclear morphology and immuno-positive staining. Total positive cells were reported as a percent of total area (pixels) in a region of interest that excluded scab, epidermis, and cartilage plate. The ID of the samples being run was blinded from the user until the end of the analysis. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens, Acomys cahirinus