Journal: Frontiers in Immunology

Article Title: Enhanced Susceptibility of ADAP-Deficient Mice to Listeria monocytogenes Infection Is Associated With an Altered Phagocyte Phenotype and Function

doi: 10.3389/fimmu.2021.724855

Figure Lengend Snippet: Distinct changes in NET formation by neutrophils from ADAP-deficient mice in response to in vivo Listeria monocytogenes ( Lm ) infection. Wild type (▪) and ADAPko (▫) mice (age: 10–14 weeks) were infected i.v. with 2.5 × 10 4 CFU Lm (strain 10403S) and sacrificed at the indicated times post infection. Formalin-fixed tissue slices were stained with primary antibody (neutrophil elastase (NE) and histone H3) and subsequently with the secondary antibody (Alexa Fluor 488 donkey anti-rabbit IgG and goat anti-rat IgG Alexa Fluor 647) as well as DAPI for nuclei staining. Four pictures per slide (three different layers) at a magnification of ×200 were taken, and histones were counted using Image-Pro Plus 6 (double-blinded). Representative pictures show the overlay of the nuclei (colored in blue), histones (colored in red), and neutrophil elastase (colored in green) in (A) spleen and (B) liver tissues, whereas white arrows highlight the histones. Summary plots show the means of counted histones. Data are depicted as box and whiskers ± min to max for n = 4 individually analyzed mice per genotype out of one experiment. Statistical analyses were performed using two-way ANOVA with Bonferroni’s post-hoc test (*p < 0.05). NET, neutrophil extracellular trap; ADAP, adhesion and degranulation-promoting adaptor protein; ADAPko, adhesion and degranulation-promoting adaptor protein knockout.

Article Snippet: The same procedure was followed for the secondary antibodies conjugated to Alexa Fluor 488 donkey anti-rabbit IgG (Bioss) and Alexa Fluor 647 goat anti-rat IgG (Thermo Fisher) with an incubation of 30 min at RT in the dark.

Techniques: In Vivo, Infection, Staining, Knock-Out