mouse monoclonal antibody against human muc5b  (Santa Cruz Biotechnology)

Journal: Stem Cell Research & Therapy

Article Title: Salivary gland organoid transplantation as a therapeutic option for radiation-induced xerostomia

doi: 10.1186/s13287-024-03833-x

Figure Lengend Snippet: Generation and characterization of collagen-based hSGOs. ( A ) Microscopic image of the growth of hSGOs cultured in a type I collagen matrix. Scale bar, 100 μm. ( B ) Organoid diameters at days 4 and 7 of passages 1 to 4. ( C ) Cumulative cell number of passage 1 to 8. n = 3, biologically independent samples. ( D ) Gene expression of the acinar cell markers ( AMY1A , AQP5 , and CRISP3 ) and ductal cell markers ( KRT19 , KRT7 , and WFDC2) of undifferentiated and differentiated organoids. n = 3 or 4, biologically independent samples. * p < 0.05. ( E ) Immunofluorescence staining for acinar cell markers (AQP5, AMY, CD166, and MUC5B) and ductal cell markers (KRT7 and KRT19) in undifferentiated and differentiated hSGOs and tissue. Scale bar, 100 μm. ( F ) H&E and PAS staining in undifferentiated and differentiated hSGOs and tissue. Scale bar, 100 μm. ( G ) Measurement of amylase activity of undifferentiated and differentiated hSGOs. n = 4, biologically independent samples. *** p < 0.001

Article Snippet: The primary antibodies used for staining were: mouse anti-cytokeratin 19 (1:200, Biolegend); rabbit anti-cytokeratin 7 (Roche); mouse anti-α amylase (1:200, Santa cruz); rabbit anti-aquaporin 5 (1:200, Alomone labs); mouse anti-MUC5B (1:200, Abcam); rabbit anti-CD166 (1:200, Novus Biologicals); rabbit anti-Ki67 (1:300, Abcam); mouse anti-cytokeratin 17 (1:200, Santa cruz); mouse anti-cytokeratin 14 (1:200, Abcam); mouse anti-human nucleoli (1:100, Abcam); rabbit anti-E-cadherin (1:300, Abcam).

Techniques: Cell Culture, Expressing, Immunofluorescence, Staining, Activity Assay

Journal: Stem Cell Research & Therapy

Article Title: Salivary gland organoid transplantation as a therapeutic option for radiation-induced xerostomia

doi: 10.1186/s13287-024-03833-x

Figure Lengend Snippet: Transplantation of collagen-based hSGOs in a xerostomia mouse model. ( A ) Strategy for transplantation of hSGOs to a radiation-induced xerostomia mouse model. Scale bar, 250 μm. ( B ) Engrafted region of parotid gland organoids labeled with Cytopainter at 2 and 4 weeks (Left, white arrowhead indicate an engrafted region). Historical analysis revealed large nuclei-consisting organoids in the recipient’s submandibular gland at 4 weeks post-transplantation (Right, yellow dashed line). Scale bar, 100 μm. *** p < 0.001. ( C ) Human E-cadherin, human nucleoli, KRT19, CD166 were detected in the engrafted organoid of the recipient’s submandibular gland by immunofluorescence staining. Scale bar, 100 μm. ( D ) Immunofluorescence staining with MUC5B and Alcian blue staining for mucin secretion. Scale bar, 100 μm. ( E ) Historical analysis of integrated organoids on 16 weeks post-transplantation (Yellow dashed line). Scale bar, 100 μm. ( F ) Expression of human E-cadherin, KRT19, and ( G ) MUC5B in engrafted hSGOs at 16 weeks post-transplantation. Scale bar, 100 μm. ( H ) Alcian blue staining for mucin secretion in engrafted hSGOs at 16 weeks post-transplantation. Scale bar, 100 μm

Article Snippet: The primary antibodies used for staining were: mouse anti-cytokeratin 19 (1:200, Biolegend); rabbit anti-cytokeratin 7 (Roche); mouse anti-α amylase (1:200, Santa cruz); rabbit anti-aquaporin 5 (1:200, Alomone labs); mouse anti-MUC5B (1:200, Abcam); rabbit anti-CD166 (1:200, Novus Biologicals); rabbit anti-Ki67 (1:300, Abcam); mouse anti-cytokeratin 17 (1:200, Santa cruz); mouse anti-cytokeratin 14 (1:200, Abcam); mouse anti-human nucleoli (1:100, Abcam); rabbit anti-E-cadherin (1:300, Abcam).

Techniques: Transplantation Assay, Labeling, Immunofluorescence, 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

Journal: International Journal of Pharmaceutics: X

Article Title: Impact of mucus modulation by N -acetylcysteine on nanoparticle toxicity

doi: 10.1016/j.ijpx.2023.100212

Figure Lengend Snippet: Calu-3 cells cultured under AIC conditions secrete the major gel forming mucins MUC5AC and MUC5B. A-C) 2D sections taken above the cells demonstrate the secretion of both MUC5AC and MUC5B. D-E) 2D sections through the cells show the presence of MUC5AC and MUC5B filled granules. Samples were stained with anti-MUC5AC Alexa Fluor 488 (green) and anti-MUC5B-Atto 647 N (magenta). Scale bar 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For MUC5B staining, samples were incubated with 2 μg/ml mouse anti-MUC5B primary antibody (Thermo Fisher Scientific, Rockford, USA) for 1 h at RT and 10 μg/ml Atto 647 N conjugated anti-mouse secondary antibody (Sigma Aldrich, Siegen, Germany) for 1 h at RT.

Techniques: Cell Culture, Staining