Computational protocol: In Vivo Non-Invasive Tracking of Macrophage Recruitment to Experimental Stroke

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Protocol publication

[…] For PB staining cells were fixed in 4% PFA for 15 min at room temperature. After washing, cells were incubated for 30 min with staining solution (1:1 2% hydrochloric acid (HCl), 2% potassium ferrocyanide [K4Fe(CN)6]). Following PB exposure cells were rinsed with distilled water and coverslipped with Aqua Poly/Mount (Polysciences Inc., Warrington, PA, USA).PB staining of tissue sections was conducted with initial incubation for 10 min with 10% K4Fe(CN)6 followed by 30 min incubation with staining solution (1:1 4% HCl, 4% [K4Fe(CN)6]). The tissue sections were washed for 5 minutes with tap water, followed by counterstaining of cell nuclei with nuclear fast red for 10 seconds.For immunohistochemical (IHC) stainings of tissue sections mice were deeply anesthetized with isoflurane and transcardially perfused with 2xPBS followed by 4% PFA. Brains were dissected from skull and post-fixed overnight, before transfer into 30% sucrose solution. Once sunk to the vial’s bottom, brains were frozen in -60°C cold 2-methylbutane and stored at -80°C. 14 μm thin tissue sections of the graft area and 20 μm thin tissue sections of the ischemic lesion site, respectively, were cut in coronal plane using a cryostat (Leica, Wetzlar, Germany) and stored at -20°C.For immunocytochemistry (ICC), cells were fixed with 4% PFA for 10 min at room temperature. In antibodies used for ICC/ IHC are given. For both, IHC and ICC, a nuclei stain was performed (Hoechst Dye 1:1000; Hoechst).All stainings of cell and tissue sections were microscopically analysed. For brightfield (BF), phase contrast (PC), as well as fluorescence images of cells and tissue sections, a fluorescent microscope (Keyence BZ-9000) was used with 4x, 20x, 40x, and 60x magnification. Fluorescence 3D-stack of tissue sections was acquired using a confocal microscope (Leica TCS SP8). Here, 20x magnification was chosen. The recorded images were preprocessed using the manufacturers’ software (BZ-II Analyzer 2.1 for the Keyence microscope, Las X Software for confocal microscope). Brightness and contrast were finally adjusted for each experimental setup using ImageJ software (Version 1.48g). [...] All data are presented as mean ± standard deviation. Comparison of means of two independent groups was conducted using Mann-Whitney test for non parametric data distribution. Multiple comparisons were conducted using either one way ANOVA with Tukey’s post-test (for normal data distribution), or Kruskal-Wallis test followed by Dunn’s correction for non parametric data (Graph Pad Prism version 6.01, GraphPad Software, Inc., San Diego, USA). Longitudinal in vivo BLI data was analysed using a repeated measures ANOVA with Bonferroni correction (SPSS version 22, IBM SPSS statistics, Ehningen, Germany). If not specified differently, a p-value ≤ 0.05 was considered significant and highlighted by */#, in addition a p ≤ 0.005 was marked by ** and p ≤ 0.001 is represented by ***/###. […]

Pipeline specifications

Software tools LAS X, ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Stroke