Computational protocol: Anti-inflammatory Microglia/Macrophages As a Potential Therapeutic Target in Brain Metastasis

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Protocol publication

[…] Immunohistochemistry was performed on 10 µm thick tissue sections mounted on glass slides (Superfrost Plus, Menzel Gläzer, Braunschweig, Germany). Initially, slides were allowed to come to room temperature before rehydration in PBS (Thermo Fisher Scientific, UK; pH 7.4). When necessary, antigen retrieval was performed using sodium citrate buffer containing 0.1% Tween 20 (pH 6.0). Tissue was permeabilized in PBS containing 0.05% Tween 20 and endogenous peroxidases were quenched in 1% (v/v) hydrogen peroxide (30% w/w) (Sigma, Aldrich) in methanol. Brain sections were blocked for non-specific antibody binding in 10% serum (in PBS) for an hour and incubated overnight at 4°C with primary antibodies (1% serum in PBS): anti-Iba1 (Abcam ab5076), anti-iNOS (M-19) (SantaCruz sc650), anti-Arg1 (Novus Biologicals NBP1-54621), anti-MRC1 (AbD Serotec MCA2235), anti-COX2 (Abcam ab15191), anti-CC3 (Asp175) (Cell Signaling Technology 9661), and anti-CD31 (R&D AF3628). After rinsing in PBS, tissue sections were incubated with appropriate secondary antibodies (Vector Labs, CA, USA) for an hour: biotinylated horse anti-goat IgG, biotinylated goat anti-rabbit IgG, biotinylated rabbit anti-rat (mouse absorbed) IgG. This step was followed by incubation with the avidin/biotin complex-horseradish peroxidase (HRP) system (VECTASTAIN Elite ABC Kit Standard, Vector Laboratories, CA, USA). Peroxidase was detected using 3,3′-diaminobenzidine (DAB) and tissue was counterstained with 0.5% cresyl violet. Tissue was dehydrated using increasing concentration of ethanol, cleared in xylene and mounted in DPX mountant (Fisher Scientific, UK). Immunostained slides were imaged on ScanScope CS slide scanner (Aperio, Vista, CA, USA) and analyzed using ImageScope (Aperio, USA).Quantitative analysis of the volumes of metastatic areas and microglial/macrophage infiltration was performed by manual demarcation of the areas of interest on brain sections 50 µm apart spanning the metastatic lesions: tumors were defined as cresyl violet-positive foci and microglial/macrophage infiltration area by the outer limit of reactive Iba1+ cells. Quantitation of DAB for inflammatory markers was performed using the Positive Pixel Count Algorithm (Aperio, USA) and converted to area (μm2) of immunostaining. Positive and strong positive pixels within the areas of interest were included for the analysis. In the IL-4 in vivo study, numbers of Iba1+ cells were quantified across different fields of view from multiple sections, as specified. [...] An in-house Matlab/ImageJ based approach to coregister immunostains on sequential sections was developed. 10 µm thick sequential brain sections from mice bearing 4T1-GFP metastases were stained for different biomarkers: Iba1 for microglia/macrophage; iNOS and COX2 for proinflammatory phenotype; Arg1 and MRC1 for anti-inflammatory phenotype and counterstained with cresyl violet. Sequential IHC images were coregistered by choosing landmark points that correspond to the same areas on both images based on metastatic lesion and brain morphology (corpus callosum, ventricle). Coregistered images were subsequently thresholded by color to extract maps of the different stains and merged into a single overlap map. The overlap maps show colocalization of Iba1 with either polarization marker in white, and immunostained pixels for the polarization markers that did not colocalize with Iba1 pixels on the sequential section in red. The white pixels (double labeling) corresponded to the immunostained object for the polarization markers, rather the microglia/macrophage marker, to yield a percentage of Iba1-positive pixels that were positive for each of the polarization markers. [...] Immunofluorescent detection of antigens was performed on 10–20 µm thick tissue sections mounted on glass slides (Superfrost Plus, Menzel Gläzer, Braunschweig, Germany). Tissue was permeabilized with PBS Tween 20 (0.05%), endogenous peroxidase activity was quenched in 1% H2O2 in PBS and endogenous biotin was blocked using the streptavidin/biotin blocking kit (Vector Laboratories, CA, USA). Tissue was blocked in TNB buffer (PerkinElmer, UK) for 1 h and primary antibodies were applied overnight at 4°C: anti-Iba1 (Abcam ab5076), anti-iNOS (M-20) (sc651), anti-Arg1(H-52 sc20150), anti-MRC1 (AbD MCA2235), anti-CC3 (Asp175) (CST 9661), and anti-GFP (Abcam ab13970). Next day, sections were washed in PBS and incubated with the appropriate fluorophore conjugated secondary antibodies for an hour: anti-goat TexasRed or antigoat DyLight 594 (Vector Labs, CA, USA), anti-rabbit TexasRed (Vector Labs, CA, USA), and anti-chicken CFTM488A (Sigma-Aldrich, UK). Alternatively, biotinylated secondary antibodies were applied on sections for 1 h followed by streptavidin-conjugated fluorophores (AMCA or Dylight 488) (Vector Labs, CA, USA) for an additional hour. iNOS and Arg1 signals were amplified using the TSA Biotin System (PerkinElmer LAS, UK). Briefly, after the incubation with anti-rabbit biotinylated secondary antibody, streptavidin–HRP (1:200 in TNB buffer) was applied to the tissue for 30 min followed by a 10 min incubation with biotinylated TSA (1:100 in amplification buffer). Streptavidin-conjugated AMCA was used as the final step for the amplification protocol.A colocalization analysis was performed between the anti-inflammatory marker MRC1 and Iba1 using an in-house ImageJ plugin. The parameters measured by the plugin were the percentage area of marker A’s signal that is above a user set threshold, the percentage area of marker B’s signal that is above a user set threshold, the percentage area of the image where both markers are above the user set threshold (the colocalized image area), and the percentage of each marker’s signal that has overlaps with the colocalized image area. The plugin also calculated the Pearson’s correlation coefficient and the Mander’s overlap coefficient.Immunofluorescent staining was also performed on cells grown on coverslips and treated appropriately (see below). Cells were fixed in 4% PFA (in PBS, pH 7.2) for 10 min and washed in PBS twice. Cells were permeabilized with PBS Tween 20 (0.05%) and blocked for endogenous biotin using the streptavidin/biotin blocking kit (Vector Laboratories, CA, USA). Subsequently, cells were incubated with TNB buffer (PerkinElmer) for 1 h in a humidified chamber to block non-specific antibody binding. Appropriate primary antibodies were applied to the cells overnight at 4°C: anti-rat MRC1 (AbD MCA2235), anti-mouse F4/80 Biotin (eBioscience, 13-4801-81), anti-iNOS (M-20) (sc651). Next day, cells were washed in PBS and appropriate fluorophore-conjugated fluorophores (anti-rat Texas Red, anti-rabbit Texas Red, streptavidin-conjugated Dylight 488) were applied to the cells for 1 h at room temperature. Cell nuclei were counterstain with DAPI (Vector Laboratories, CA, USA). […]

Pipeline specifications

Software tools ImageScope, ImageJ, Coloc
Applications Whole slide imaging, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Brain Diseases, Breast Neoplasms, Carcinoma, Neoplasms
Chemicals Mannose, Nitric Oxide