Journal: bioRxiv

Article Title: Trickle infection and immunity to Trichuris muris

doi: 10.1101/677096

Figure Lengend Snippet: (A) Relative expression of goblet cell secreted products measured by qPCR. (B) Western blot to visualize Muc5ac protein from extracted mucus from naïve or T. muris infected C57Bl/6 mice at 11 weeks p.i.. (C) Muc2+ goblet cells per crypt and representative immunostained sections during trickle infection. Muc2 in green. DAPI stain in blue. n=5, statistical analysis completed by a one way-ANOVA comparing each time point to week 0. Data presented as mean +/- SEM, *=p<0.05.

Article Snippet: Mucins were transferred onto a nitrocellular membrane, blocked with casein and probed using chicken anti-mouse Muc5ac antibody (Rockland) followed by an incubation with the goat anti-chicken IgY AF790 (Abcam).

Techniques: Expressing, Western Blot, Infection, Staining

Journal: bioRxiv

Article Title: Trickle infection and immunity to Trichuris muris

doi: 10.1101/677096

Figure Lengend Snippet: Following CD4+ T cell depletion worm expulsion mechanism were analyzed. (A) Representative images of PAS stained caecal sections to measure crypt length and goblet cell counts per crypt, n=5. (B) Relative expression of Muc5ac and RELM-beta analyzed by qPCR, n=5. (C) Epithelial cell turn over measured by furthest distanced BrdU stained cells travelled up intestinal crypt. Sections stained by anti-BrdU (green) and DAPI (blue), n=4-5. Statistics measured using an unpaired t test. Data presented as mean +/- SEM, *=p<0.05, **=p<0.01.

Article Snippet: Mucins were transferred onto a nitrocellular membrane, blocked with casein and probed using chicken anti-mouse Muc5ac antibody (Rockland) followed by an incubation with the goat anti-chicken IgY AF790 (Abcam).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Requirement for Fucosyltransferase 2 in Allergic Airway Hyperreactivity and Mucus Obstruction

doi: 10.1101/2024.05.10.593580

Figure Lengend Snippet: A . α-Fuc was detected using the lectin Ulex europaeus agglutinin-1 (UEA1, yellow, 8 μg/ml), which labeled airway mucous cells and a mucus plug in a person who died from fatal asthma. The airway mucins MUC5AC (magenta, 1:1,000) and MUC5B (cyan, 1:500) were both detected. B-F . UEA1 labels mouse airway epithelium after induction of allergic inflammation using Aspergillus oryzae extract (AOE), and α-Fuc detection is Fut2-dependent. In C57BL/6 mice exposed to AOE ( B ), MUC5B (cyan, 1:500), MUC5AC (magenta, 1:2,500) and α-Fuc (UEA1, yellow, 8 μg/ml) labeling co-localize within airway epithelia. In Fut2 +/+ mice at baseline ( C ), α-Fuc and Muc5ac are not detectable in airway epithelia, but Muc5b is observed. After AOE, Muc5b is sustained while Muc5ac and α-Fuc detection are both induced in Fut2 +/+ animals ( D ). In Fut2 −/− mice, baseline and AOE-induced expression of Muc5b ( E ) and Muc5ac ( F ) are sustained, but AOE-induced α-Fuc detection is lost ( F ). Nuclei are stained with DAPI (blue). Scale bars: 100 μm for low magnification images and 50 μm for insets in A and C-F ; 10 μm for low magnification image and 8 μm for insets in B .

Article Snippet: Mouse mucins were detected using polyclonal rabbit-anti-mouse MUC5AC produced against the peptide N-CHALGDTSHAESSEQEFKSKESEEHGQQLAFR (Pacific Immunology, Ramona, CA, 1:2500 dilution), mouse-anti-mouse MUC5B (clone MDA-3E1, Cat. MABT899, MilliporeSigma, Burlington, MA, 1:500 dilution), and UEA1 (as above).

Techniques: Labeling, Expressing, Staining

Journal: bioRxiv

Article Title: Requirement for Fucosyltransferase 2 in Allergic Airway Hyperreactivity and Mucus Obstruction

doi: 10.1101/2024.05.10.593580

Figure Lengend Snippet: Lung lavage fluid was obtained from saline and AOE challenged Fut2 +/+ (black) and Fut2 −/− (magenta) mice. A , B . AOE challenge resulted in increases in total numbers of cells ( A ) and eosinophils ( B ), which did not differ between Fut2 +/+ and Fut2 −/− mice. Data were compared using groups by Kruskal-Wallis ANOVA. ‘*’, p < 0.05 using Dunn’s post-hoc test for multiple comparisons between genotype and challenge groups (p-values are shown in A and B ). Comparisons were made between AOE challenged Fut2 +/+ mice (n = 9 biological replicates), AOE challenged Fut2 −/− mice (magenta, n = 10 biological replicates), saline challenged Fut2 +/+ mice (n = 10 biological replicates), and saline challenged Fut2 −/− mice (magenta dashed lines, n = 9 biological replicates). Solid lines and filled circles identify AOE challenged animals; dashed lines and open circles depict saline challenged controls. C . Combined immuno- and lectin blotting was performed on neat lavage. Equal volumes of lavage fluid (27 μl) were loaded per well, separated in 1% SDS agarose gels, and transferred to PVDF membranes. Membranes were probed with biotinylated UEA1 (1:1,000, 2 μg/ml) and either rabbit-anti-MUC5AC (1:2,000) or rabbit-anti-MUC5B (1:5,000). For secondary detection, Alexa 680-conjugated streptavidin and Alexa 800-conjugated labeled goat-anti-rabbit probes (Licor, 1:15,000) were applied. Monochrome images were acquired and pseudocolored magenta (MUC5AC), cyan (MUC5B), or yellow (UEA1). Image overlays demonstrate UEA and mucin signal colocalization (white in merged panels).

Article Snippet: Mouse mucins were detected using polyclonal rabbit-anti-mouse MUC5AC produced against the peptide N-CHALGDTSHAESSEQEFKSKESEEHGQQLAFR (Pacific Immunology, Ramona, CA, 1:2500 dilution), mouse-anti-mouse MUC5B (clone MDA-3E1, Cat. MABT899, MilliporeSigma, Burlington, MA, 1:500 dilution), and UEA1 (as above).

Techniques: Saline, Labeling