MA5-12178

antibody from Invitrogen Antibodies

Targeting: MUC5AC MUC5

Provider product page for MA5-12178

Immunocytochemistry

Immunohistochemistry

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Antibody data

Antibody Data
Antigen structure
References [278]
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Validations
Immunocytochemistry [1]
Immunohistochemistry [6]
Other assay [34]

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Product number: MA5-12178 - Provider product page
Provider: Invitrogen Antibodies
Product name: MUC5AC Monoclonal Antibody (45M1)
Antibody type: Monoclonal
Antigen: Other
Description: MA5-12178 targets Mucin 5AC in IHC (P) and ICC/IF applications and shows reactivity with Chicken, Feline, Human, mouse, Non-human primate, Porcine, Rabbit, Rodent, and Rat samples. The MA5-12178 immunogen is m1 mucin preparation from the fluid of an ovarian mucinous cyst belonging to an O Le(a-b-) patient.
Reactivity: Human, Mouse, Rat, Chicken/Avian, Feline, Porcine, Rabbit
Host: Mouse
Isotype: IgG
Antibody clone number: 45M1
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

MUC5AC protein structure - MA5-12178 shown in red.

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Experimental details: Formalin-fixed, paraffin-embedded human stomach stained with Mucin 5AC antibody using peroxidase-conjugate and DAB chromogen. Note staining of extracellular mucins in the surface of the stomach.

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Experimental details: Formalin-fixed, paraffin-embedded rat stomach stained with Mucin 5AC antibody using peroxidase-conjugate and DAB chromogen. Note cytoplasmic and surface staining of mucous cells.

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Experimental details: Formalin-fixed, paraffin-embedded human stomach stained with Mucin 5AC antibody using peroxidase-conjugate and DAB chromogen. Note staining of extracellular mucins in the surface of the stomach.

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Experimental details: Formalin-fixed, paraffin-embedded rat stomach stained with Mucin 5AC antibody using peroxidase-conjugate and DAB chromogen. Note cytoplasmic and surface staining of mucous cells.

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Experimental details: Immunofluorescent analysis of Mucin 5AC (green) showing staining in the cytoplasm of human stomach tissue (right) compared to a negative control without primary antibody (left). Formalin-fixed tissue was permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Tissue was probed with a Mucin 5AC monoclonal antibody (Product # MA5-12178) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Tissue was washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

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Experimental details: Formalin-fixed, paraffin-embedded rat stomach stained with Mucin 5AC antibody using peroxidase-conjugate and DAB chromogen. Note cytoplasmic and surface staining of mucous cells.

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Experimental details: NULL

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Experimental details: Fig. 7 Representative immunohistochemistry images (alcian blue + H&E, MUC-5AC and FOX-J1) of 3D lung models after 28 days of repeated exposure to 30, 60 or 90 puffs of myblu aerosol (undiluted), 3R4F cigarette smoke (dilution 1:17 smoke: air) or negative control (90 puffs of humidified filtered air). Tissues exposed to IL-13 was evaluated at T19. x in table (Quantified staining average). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Experimental details: Image 1 Expression of MUC5AC and TFF1 in normal colon ( A ), hyperplastic polyps (HPs) ( B ), and sessile serrated adenomas/polyps (SSA/Ps) ( C ) (x10). Immunofluorescence and immunoperoxidase staining of the same region is shown for representative control (normal), HP, and SSA/P samples. As expected, the signals for gastric proteins MUC5AC and TFF1 were negligible in normal colonic mucosa. MUC5AC and TFF1 immunopositive cells were present in HPs. In SSA/Ps, MUC5AC and TFF1 were typically expressed by most of the epithelial cells along the entire length of architecturally compromised crypts. SSA/Ps, compared with HPs, also showed significant coexpression of both MUC5AC and TFF1 in merged immunofluorescence analyses.

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Experimental details: 3 FIGURE Tracheal mucin staining and bronchoalveolar concentrations in pigs challenged with bethanechol. (a,b) Representative antibody labelling of MUC5AC (green) and MUC5B (red) in 10-um-thick tracheal cross-sections from piglets challenged with intra-airway saline (a) or intra-airway bethanechol (b). Nuclei are shown in blue (Hoechst). Arrows indicate examples of surface mucus and asterisks examples of submucosal glands. (c-f) MUC5AC and MUC5B staining signal intensity of the surface epithelia (c,d) and submucosal glands (e,f). (g,h) Bronchoalveolar lavage fluid concentrations of MUC5AC (g) and MUC5B (h). For panels c-f, n = 20 saline-challenged piglets (10 females and 10 males) and n = 20 bethanechol-challenged pigs (10 females and 10 males). For panels g and h, n = 11 saline-challenged pigs (five females and six males) and n = 11 bethanechol-challenged pigs (five females and six males). Data points represent the mean fluorescence intensity for each piglet calculated from three to five images analysed (encompassing the anterior, lateral and posterior regions of the trachea). All data are shown as mean values +- SD

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Experimental details: FIG. 2. Immunofluorescent staining of vimentin, CK5, CK18, MUC5AC, and MUC5B in 3D airway mucosa tissue models. Vimentin is localized in the SIS of each cell line-based model (A, E, I, M) . 3D tissue models based on HBEC3-KT, VA10, and Cl-huAEC show CK5 ( red ) and CK18 ( green ) expression in the epithelial compartment (B, F, N) , whereas Calu-3-based models show only CK18 expression (J) . MUC5AC and MUC5B are identified in HBEC3-KT- (C, D) , VA10- (G, H), and Calu-3-based models (K, L), but are not visible in Cl-huAEC-based models (O, P) . Scale bars: 50 mum. CK, cytokeratin; MUC, mucin; SIS, small intestinal submucosa.

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Experimental details: Figure 3 Mucus characteristics in HNE organoids, representative findings from CF and non-CF organoids are shown. ( A ) MUC5B granules in G551D/N1303K organoid. Scale = 50 um. ( B ) Muc5B whole-mount IF showing a bundled mucin pattern in F508del/R117H-5T organoid. Scale = 50 um. ( C ) MUC5AC fixed cross-section showing a diffuse mucin pattern in a non-CF organoid. Scale = 50 um. ( D ) Top panel is the maximum projection of the 3-D hemisphere in the bottom panel. MUC5B is seen entering the lumen (arrow) through the apical surface in a non-CF organoid. Scale = 500 um. ( E ) Brightfield microscopy showing a clump of cellular debris and/or mucus in the lumen in a non-CF organoid. Scale = 100 um. ( F ) uOCT still of probable mucus in a non-CF organoid. ( E , F ) correspond to Videos S1 and S2 . E = epithelium L = lumen M = mucus.

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Experimental details: Figure 1 Characterization of wdNHBE cells by histology and immunocytochemistry. ( a ) Hematoxylin and eosin staining of a transverse section of NHBE cells differentiated at the air liquid interface on the apical surface of a transwell membrane. Nuclei ( n ) are stained purple and membranes, cytoplasm and cilia are stained pink. Basal cells ( b ), ciliated cells ( c ) with surface cilia, goblet cells ( g ) and the porous polyester transwell membrane ( m ). ( b ) En face images of wdNHBE cells stained with MUC5AC (green, goblet cells) and MUC5B (red, secretory cells). The Merge of MUC5AC, MUC5B and phalloidin (F-actin)-stained cytoskeleton (pink) depicts the partial colocalization of MUC5AC and MUC5B secretion (yellow, indicated by arrows). En face Z-stacks of 19 slices (9 um) were captured using a 40x oil immersion objective and maximum intensity projections generated, 20 um scale bars indicate cell size. ( c ) Confocal microscopy of wdNHBE cells stained with acetylated tubulin (TubAc, green, cilia on the surface of ciliated cells) and CLDN4 (red, tight junctions). Merge of TubAc, CLDN4 and F-actin. Maximum intensity projections of en face Z-stacks, 19 slices (9 um) imaged using a 40x oil immersion objective with 20 um scale bars are shown.

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Experimental details: FIGURE 1 Overexpression of Substance P increases MUC5AC. (a) Overview of the experimental model. (b) Concentrations of SP in lung slice culture media 48 h post-AAV transduction ( n = 12; 6 males, 6 females). (c) Antibody-labeling of mucin 5AC (MUC5AC; red) and (d) MUC5AC signal intensity quantification ( n = 12; 6 males, 6 females). (e) Antibody-labeling of mucin 5B (MUC5B; red) and (f) MUC5B signal intensity quantification ( n = 11; 6 females, 5 males). (g) MUC5AC protein (red) in vehicle and SP-treated cultured swine airway epithelia and (h) quantification of MUC5AC fluorescence intensity ( n = 6 individual cultures from six unique piglets; three females, three males). Nuclei are shown in blue (Hoechst). Groups were compared by paired T -test. Data are shown as arbitrary units (A.U.). AAV, Adeno-associated virus; GFP, green fluorescent protein; OE, overexpression; SP, Substance P. Lines show matched pairs. Mean, standard error of the mean (SEM), Log2 Fold-change, and p -values are shown in Tables S1-S2.

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Experimental details: Figure 7 iEVs strongly enhance Mucin 5ac in mice with colitis. (A) Quantification of MUC5ac in colon mucosa in nMSCs and iMSCs mice: IF (left), quantification (right). (B) Quantification of mucin 5ac in colon mucosa in nEVs and iEVs mice: IF (left), quantification (right). Results are mean +- standard error (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

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Experimental details: Figure 2 Atg16L1 deficient mice have increased intracellular mucins and normal stimulated mucin secretion. ( A , B ) Representative immunoblots of lung homogenates for Muc5b and Muc5ac by lectin UEA-1 from OVA challenged WT or Atg16L1 HM mice with corresponding quantification normalized to total protein level (n = 8 WT mice and 9 Atg16L1 HM mice). Dashed box denotes area of mucin quantification. ( C ) Representative images of mucin immunostaining of airways by mouse anti-Muc5b and Muc5ac by lectin UEA-1 in OVA-challenged Atg16L1 HM and WT mice +- ATP nebulization with corresponding quantification of mucin levels (nl/mm 2 ) for Muc5b ( D ) and UEA-1/Muc5ac ( E ) (n = 6 mice per group). Arrows point to mucous cells that have recently emptied intracellular mucin while arrow heads show non secreted mucous cells. Scale bar = 50 microns. Low power magnification representative images ( F ) with * showing mucous plugging in the airways. Arrow heads show intracellular mucins remaining in the epithelial secretory cells. Scale bar = 100 microns. Graphs show scatter plots with median bar for mucin immunostaining and scatter plots with median and interquartile range distribution bars for mucin blots. Significant difference by ANOVA with Tukey's post-hoc comparisons with * for ATP treatment difference and ** for Atg16LHM difference for staining in parts ( C ) and ( D,E ) and Mann-Whitney test for mucin western blots in parts ( A ) and ( B ).

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Experimental details: Figure 5 Atg16L1 deficient mice have slower resolution of mucous metaplasia following Type 2 airway inflammation. ( A ) Muc5ac and Muc5b expression levels were measured by qRT-PCR from a representative WT mouse lung homogenate in a time course experiment at 0, 3, 10, 17, and 24 days after the last OVA treatment. Data reported as fold change vs. non-treated mice (N = 4 for timepoints 0, 3, and 10 and n = 1 mouse at day 17 and 24). (B ) Representative Lc3 immunoblot from whole lung homogenates (Lh) or after lysosome isolation (Ly) was performed from WT mouse lung homogenates +- OVA sensitization and challenge and then day 0, 3 and 10 from last OVA challenge with total protein stain shown below. (n = 2 per group) ( C ) Cathepsin B proteolytic enzyme activity was measured from isolated lysosome fractions (Ly) from mouse lung homogenates +- OVA. Enzyme activity normalized by total protein (n = 2 per group for naive mice and n = 6 per group at day 0 and 7 per group at days 3 and 10 after OVA. ( D ) Representative Lc3 and Sqstm1 immunoblots from WT and Atg16L1 HM mouse lung lysosomes at day 0,3, and 10 from last OVA challenge. Quantification of Lc3 II ( E ) and Sqstm1 ( F ) according to time from last OVA challenge from WT and Atg16L1 HM lysosomal isolates (N = 7 mice per group). ( G , H ) Representative immunoblots of Muc5b and Muc5ac by lectin Uea-1 from WT and Atg16L1 HM mouse lung homogenates at 10 days after last OVA challenge with corresponding quantification. Mucin band densi

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Experimental details: Figure 6 Atg16L1 deficient mice have increased partial or incomplete fusion of Lc3, Lamp1- labeled vesicles with mucin granules at day 3 of resolution from OVA-mediated mucous metaplasia. ( A ) Lysosome were purified from WT and Atg16L1 HM mouse lungs at day 3 of resolution from OVA-mediated mucous metaplasia as described in Fig. 5 . ( B ) Representative immunoblot for Muc5ac detected by lectin UEA-1 from WT and Atg16L1 HM mouse lung lysosome preparations with corresponding quantification normalized to total protein (n = 8 mice for each group). ( C ) Representative SR-SIM images of cytospins on glass slides from lysosome preparations of WT and Atg16L1 HM mouse lungs stained by Muc5ac using lectin UEA-1 (green). (N = 7 images from 4 different mice for both WT and Atg16L1HM groups). Scale bar = 2 microns. ( D ) Representative SR-SIM images from WT and Atg16L1 HM mouse airways at day 3 of resolution from OVA-mediated mucous metaplasia with LC3 (blue), Lamp1 (red), and Muc5ac detected by lectin UEA-1 (green). Scale bar = 5 microns. Insets below show each magnified image of fusion event and have scale bar = 1 micron. Individual channels are shown below inset with gold dashed circle showing area of overlap. ( E ) Quantification fusion events among Lc3 and Lamp1 vesicles and mucin granules. N = 3 Atg16L1HM mice with 26 distinct secretory cells and 81 fusion events. N = 5 WT mice with 24 distinct secretory cells with 104 fusion events. Significant difference by Mann-Whitney * for gen

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Experimental details: Figure 7 Autophagy deficient airway epithelial cells have increased intracellular MUC5AC granules. ( A ) Schematic for targeted depletion of ATG16L1 and LC3B levels in Calu-3 cells under ALI culture conditions. Representative immunoblots for Calu-3 cells transfected with non-targeted (NT), ATG16L1 siRNA ( B ) or LC3B siRNA ( C ). ( D ) Representative immunostaining using mouse anti-MUC5AC in Calu-3 cells transfected with non-targeted control (NT), LC3B, or ATG16L1 siRNA and corresponding quantification ( E ). Scale bar = 20 microns. (n = 3 experiments with 10-20 microscopic images per sample). Significant difference by ANOVA with Tukey's post-hoc comparisons are noted by * NT vs Atg16L1 siRNA treatment difference or ** for NT vs. LC3B siRNA differences. Graphs show scatter plots with median bar for mucin immunostaining.

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Experimental details: Figure 8 Activation of autophagy leads to decreased intracellular MUC5AC granules in human airway epithelial cells. ( A ) Representative immunoblot of MUC5AC from cell homogenates from Calu-3 treated +- torin1 (10 muM) for 18 h with corresponding quantification by total protein levels (N = 7 per group). ( B ) Representative MUC5AC immunostaining in Calu-3 cells under ALI conditions +- torin1 treatment. Scale bar = 25microns. Corresponding quantification of MUC5AC staining volume density. N = 3 experiments with 10-20 microscope images taken per sample. ( C ) Representative MUC5AC immunostaining of Calu-3 transfected with NT or LC3B siRNA and treated +- Torin1 (10 muM) for 18 h with corresponding quantification ( D ) of MUC5AC volume density. Scale bar = 25microns. N = 2 experiments with 20 microscope images taken per sample. Graphs show scatter plots with median bar for mucin immunostaining and scatter plots with median bar and interquartile range for mucin blots. Significant difference by Mann-Whitney test with * for torin1 difference for MUC5AC blot in part ( A ) and unpaired T-test for MUC5AC immunostaining in part B . For quantification of parts ( C , D ) significant difference by ANOVA with Tukey's post-hoc comparisons are noted by * for torin1 treatment difference or ** and *** for siRNA differences.

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Experimental details: Figure 9 Activation of autophagy leads to decreased intracellular MUC5AC in human airway epithelial cells. ( A ) Representative immunoblot of MUC5AC from cell homogenates from human non-diseased AECs under ALI conditions treated with torin1 (10 muM) for 18 h with corresponding quantification by total protein levels (N = 5). ( B ) Representative MUC5AC immunostaining in human AECs under ALI conditions +- torin1 treatment (10 muM) for 18 h. Corresponding quantification of MUC5AC volume density. N = 2 experiments with 20 microscope images taken per sample. ( C ) Representative immunoblot for MUC5AC levels from cell lysates from human AECs under ALI condition derived from COPD explant airways. (n = 4 CTL and 6 torin1 treated inserts from 2 different lung explant donors) along with corresponding quantification to total protein levels. ( D ) Representative MUC5AC immunostaining in human COPD AECs under ALI conditions +- torin1 treatment (10 muM) for 18 h. N = 3 inserts from two different COPD donors with 12 microscope images taken per sample. Scale bar = 20 microns for ( B ) and ( D ). Graphs show scatter plots with median bar for mucin immunostaining and scatter plots with median bar and interquartile range for mucin blots. Significant difference by Mann Whitney test for mucin blot data in parts ( A ) and ( C ). Significant difference by unpaired T-test with * for torin1 difference for immunostaining data in parts ( B ) and ( D ).

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Experimental details: Fig. 7 Representative immunohistochemistry images (alcian blue + H&E, MUC-5AC and FOX-J1) of 3D lung models after 28 days of repeated exposure to 30, 60 or 90 puffs of myblu aerosol (undiluted), 3R4F cigarette smoke (dilution 1:17 smoke: air) or negative control (90 puffs of humidified filtered air). Tissues exposed to IL-13 was evaluated at T19. x in table (Quantified staining average). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Experimental details: Figure 6 Decreased expression of HEYL is associated with the impaired differentiation capacity of COPD HBECs. Normal ( n = 5 nonsmoker donors (blue circles) and n = 3 smoker donors (orange circles)) and COPD ( n = 6 donors (red circles)) HBECs were cultured on ALI for 28 days to differentiate into a mucociliary epithelium containing basal, club, goblet and ciliated cells. ( A - D ) Immunofluorescent staining and quantification of basal (KRT5, red), club (SCGB1A1, red), goblet (MUC5AC, green) and ciliated cells (acetylated tubulin, green). Nuclei are stained blue with DAPI. For each donor, the data are presented as percentage of positive cells for each cell type. Scale bar = 50 um. ( E , F ) qPCR analysis of HEYL expression as a function of time during ALI culture (days 0, 7, 14 and 28). ( E ) Data presented for individual HBEC donors at each time point. ( F ) Data presented as mean expression for normal ( n = 8) and COPD ( n = 6) HBEC donors at each time point. Error bars indicate SEM. * p < 0.05. Nonsignificant p -values also shown.

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Experimental details: Figure 8 HEYL overexpression can restore differentiation capacity to COPD HBECs in vitro. COPD HBECs ( n = 3 donors) were infected either control lentivirus (Lenti-Control) or lentivirus expressing HEYL (Lenti-HEYL) and then cultured on ALI for 28 days to differentiate into a mucociliary epithelium containing basal, club, goblet and ciliated cells. ( A ) Experimental design. ( B ) Hematoxylin and eosin (H&E) staining. Scale bar = 10 um. ( C - F ) Immunofluorescent staining and quantification of basal (KRT5, red), club (SCGB1A1, red), goblet (MUC5AC, green) and ciliated cells (acetylated tubulin, green). Nuclei are stained blue with DAPI. For each donor, the data are presented as fold-change in cell numbers compared to Lenti-Control infected cells. Scale bar = 50 um. * p < 0.05.

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Experimental details: Age-related differences in numeric densities of ciliated cells and MUC5AC expression in nasal epithelial cultures from children compared to elderly people. (A, B) Representative images of immunofluorescence of nasal epithelial cultures from healthy children and elderly people. Green: alpha-tubulin (ciliated cells), magenta: KRT5 (basal cells), yellow: MUC5AC (secretory cells), white: ZO-1 (tight junctions), and blue: Hoechst (cell nuclei). Scale bar, 100 um. (C-E) Quantification of alpha-tubulin + (ciliated) cells (C) , KRT5 + (basal) cells (D) , and MUC5AC + (secretory) cells (E) ( n = 14 and 11 individuals per group). (F) Transepithelial electrical resistance (TEER) ( n = 17 and 14 individuals per group). (G, H) Transcript levels of MUC5AC (G) and MUC5B (H) ( n = 16 and 12 individuals per group). (I) Representative MUC5AC and MUC5B Western blot. (J, K) Protein quantification of MUC5AC (J) and MUC5B (K) by densitometry ( n = 15 and 11 individuals per group). * p < 0.05 compared to children. Data are shown as mean +- S.E.M. Statistical analysis was performed with unpaired two-tailed t test in (D-F, J) , and with two-tailed Mann-Whitney test in (C, G, H, K) .

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Experimental details: Corneal de-epithelization model generates a severe and stable stem cell deficiency (A) De-epithelization was performed using a blunt spatula to scrap the entire corneal epithelium, including the limbus. Wound healing progress evaluated by fluorescein assay in wild-type (WT; n = 5) mice from day 0 until day 28. (B) Corneal opacity evaluation using a grading score (from 0 to 4; 0 = completely clear, 4 = completely opaque). (C) PAS staining for goblet cell identification in the cornea. Representative images are shown (40x).Scale bar, 50 mum. Quantification of PAS + cells was performed in the limbus and central cornea. (D) Mucin 5AC (Muc5AC) staining for goblet cell identification. Representative images are shown (20x). Scale bar, 75 mum. Quantification of Muc5AC + cells was performed in the limbus and central cornea. (E) Cytokeratin 8 (CK8) staining for conjunctival cell identification. Representative images are shown (20x). Scale bar, 75 mum. Quantification of CK8 + cells was performed in the limbus and central cornea. (F) Cytokeratin 19 (CK19) staining for conjunctival cells. Representative images are shown (20x). Scale bar, 75 mum. Quantification of CK19 + cells was performed in the limbus and central cornea. (G) Cytokeratin 13 (CK13) staining for conjunctival cells. Representative images are shown (20x). Scale bar, 75 mum. Quantification of CK13 + cells was performed in the limbus and central cornea. (H) p63 and BrdU staining for stem cell identification. Immediately after c

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Experimental details: Figure 4 SP ablation or neurokinin-1 receptor blockade reduces the number of goblet and conjunctival cells infiltrating the cornea (A) PAS staining for goblet cell identification in the cornea. Representative images are shown (40x). Scale bar, 50 mum. Quantification was performed along the entire cornea (limbus and central). (B) Muc5AC staining for goblet cell identification. Representative images are shown (20x). Scale bar, 75 mum. Quantification of Muc5AC + cells was performed in the limbus and central cornea. (C) CK8 staining for conjunctival cell identification. Representative images of the immunofluorescence are shown (20x). Scale bar, 75 mum. Quantification was performed along the entire cornea (limbus and central). (D) Cytokeratin 12 (CK12) and CK19 staining for corneal and conjunctival cells, respectively. Representative images are shown (20x). Scale bar, 75 mum. The last figure of the panel represents the merge between DAPI, CK12, and CK19. Quantification was performed along the entire cornea (limbus and central). (E) CK13 staining for conjunctival cells. Representative images are shown (20x). Scale bar, 75 mum. Quantification was performed along the entire cornea (limbus and central). Graphs show mean values +-SEM (n = 5 mice/group); statistical analysis by one-way ANOVA followed by Tukey's test: * p < 0.05, ** p < 0.01, *** p < 0.001.

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Experimental details: 6 Figure Stem cells are the primary epithelial sensors of ADP heptose and promote pro-inflammatory signaling Antrum organoids from wildtype mice were grown in full medium (FM) for 2-3 days, followed by respective medium conditions for 3-5 days (FM, -Rspo, -Rspo/-Wnt, +BMP2). 0.5 muM ADP heptose or 10 ng/ml TNF-alpha was added for 2 h to all organoid conditions prior to analysis. A Bright-field images of organoids in respective conditions before treatment with ADP heptose or TNF-alpha. Scale bars: 200 mum. B Immunofluorescence labeling for GSII and Muc5ac of organoids in respective conditions before treatment with ADP heptose or TNF-alpha. Scale bars: 50 mum. C qPCR expression level of Lgr5 in untreated organoids in respective medium conditions ( n = 4-5 biological replicates). D qPCR expression level of Gif in untreated organoids in respective medium conditions ( n = 4-5 biological replicates). E qPCR expression level of Cxcl1 , Cxcl2 , Cxcl10 , and Ccl2 in organoids after treatment with ADP heptose compared with untreated controls within the respective medium conditions ( n = 6 biological replicates per condition). F qPCR expression level of Cxcl1 , Cxcl2 , Cxcl10 , and Ccl2 in organoids after treatment with TNF-alpha compared with untreated controls within the respective medium conditions ( n = 2-3 biological replicates per condition). Data information: For (C, D, E, and F) data are mean + SD. * P < 0.05; ** P < 0.01; **** P < 0.0001 (one-way ANOVA + Tukey's multiple compar

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Experimental details: Figure 5 Rapidly Cycling Isthmus Progenitors Can Maintain Long-Term Self-Renewal Potential (A) Schematic showing the fluorescence signal associated with each phase of the cell cycle in the Rosa26-Fucci2a mouse. (B) Representative confocal images of stomach corpus gland sections (150 mum) of Rosa26-Fucci2a mice. Red, hCdt1-mCherry(30/120-G1); green, hGem(1/110-S/G2/M); grey, beta-catenin (I), Ki67 (II), HKATPase (III). Scale bars: 50 mum. (C) Venn diagram showing the overlap between genes more highly expressed in isolated proliferative isthmus cells and genes downregulated following 5-FU treatment, taken to be prospective candidate markers for proliferating isthmus cells. (D) Immunohistochemical staining for Ki67, counterstained with Mayer's hematoxylin (leftmost panel). In situ hybridization with Stmn1 anti-sense (middle panel) and Stmn1 sense negative control (rightmost panel) probes in mouse stomach corpus sections are shown. Stmn1 mRNA was restricted to the isthmus region delineated by Ki67 staining. Scale bars: 25 mum. (E) Schematic of the genetic strategy to trace Stmn1+ cell-derived progeny in the corpus gland. (F) Representative confocal images of the stomach corpus of Stmn1-CreERT2 mice. Red, Ki67; green, EGFP. Scale bars: 50 mum. (G) Representative confocal images of 150-mum-thick sections of the stomach corpus of Stmn1-CreERT2;R26R-tdTomato mice at 2 days, 2 weeks, 1 month, 3 months, and 6 months post-injection of 0.5-1 mg tamoxifen/20 g mouse body weight. Labeling

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Experimental details: Fig. 1 Shuttle peptide design and protein delivery to airway epithelia. a Amino acid sequences of shuttle peptides. Sequences aligned to highlight structural similarities. Cationic residues are highlighted in blue; hydrophobic residues are in gray. Remaining residues are in green. b Indel% in primary NK cells following Cas12a RNP delivery targeting NKG2A gene with indicated shuttle peptide ([Cas12a]: 1.33 uM; [crRNA]: 2.0 uM). Results quantified 48 h after delivery (mean +- SE; n = 3 independent experiments). c Shuttle peptide-mediated delivery of nuclear-targeted GFP protein to human airway epithelia (HAE) and tracheal explants. Left panel: Schematic of primary HAE grown at air-liquid interface. ASL: airway surface liquid. Right panel: photomicrograph of well-differentiated primary HAE culture demonstrating pseudostratified columnar epithelium. Hematoxylin and eosin stain, x40 magnification. d Efficiency of GFP delivery by different shuttle peptides. Peptides applied at four concentrations (5, 10, 15, or 20 muM); GFP concentrations 10 or 20 muM. Left graph: Cells were transduced with indicated peptide and GFP combinations and GFP quantified by fluorescence reader. Results mean +- SE; n = 4 donors. * P < 0.05, ** P < 0.005, *** P < 0.001, by one-way ANOVA with Tukey's multiple comparison test. Right panels: Representative en face views of epithelial sheets following GFP delivery at the highest peptide:GFP concentration (20 muM:20 muM), x20 magnification. Formulations were app

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Experimental details: Figure 1 VIP-assisted ALI culture generates physiologic mucus layer above intestinal epithelial cells. ( A ) Culture schematic. ( B ) Dose-dependent water secretion of VIP at 24 hours. ( C ) Immunofluorescence of monolayers differentiated under submerged and ALI conditions in the presence of VIP. Green: anti-Muc2; blue: Hoechst 33342. ( D ) Removal of hydrated mucus with forceps. ( E ) Side-view confocal micrograph showing tissues with mucus separating bacteria from epithelial cells at 0 and 6 days, respectively. Green: green fluorescent protein (GFP)-expressing Escherichia coli ; blue: Hoechst 33342. ( F ) Plot of mucus thickness vs duration of ALI. ( G ) Representative side-view confocal micrograph showing 1 mum red fluorescent beads separated from epithelial cells by mucus. ( H ) Immunofluorescence staining of paraffin-embedded, sectioned monolayers. DM, differentiation medium.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 The expression of mucin and MUC5AC in the nasal tissues of the CRSsNP and CRSwNP patients and the normal controls. PAS staining shows that mucin is highly expressed in the epithelial cells of CRS patients (arrowhead); IHC staining shows that MUC5AC is highly expressed in the epithelial cells of CRS patients (arrows). Both mucin and MUC5AC are mildly expressed in the normal controls. No significant difference in mucin or MUC5AC immunoreactivity was observed between the CRSsNP and CRSwNP patients. Statistical significance was calculated according to the one-way ANOVA. * P < 0.05 compared with the control group. Scale bars: 50 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 The co-expression of IL-19 and MUC5AC in the nasal tissues from the control and CRSwNP patients. Immunohistochemistry (Frozen Sections) shows that IL-19 (green) and MUC5AC (red) are highly expressed in the epithelial cells of control and CRSwNP patients. Nuclei are stained with DAPI (blue). Scale bars: 50 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. peBCs Achieve Robust Proximal Epithelial Differentiation in 3 Platforms and More Distal Character in Engineered Lung (A-D) peBC differentiated as (A) organoid, (B) ALI, (C) engineered trachea, and (D) engineered lung. (A) Bright field of organoids at 2 weeks in Matrigel (scale bar, 100 mum). (B) SEM of ALI at 3 weeks (scale bar, 10 mum). (C) H&E of engineered trachea at 4 weeks (scale bar, 50 mum). (D) Stitched H&E image and inset of the upper right lobe of engineered lung at 1 week taken with EVOS system (scale bars 750 mum, 50 mum, resp. ); IF of KRT5/ MUC5AC in alveolar region; KRT5/MUC5AC and KRT5/Tub1alpha in the trachea showing a lack of secretory and ciliated cells (scale bars, 25 mum); IF of AQP5 in alveolar region, CCSP in bronchiole (scale bars, 50 mum). In (A)-(C), IF of KRT5/MUC5AC and KRT5/Tub1alpha in organoid, ALI and engineered trachea; organoid lumens and apical cell surfaces oriented up (scale bars, 25 mum). See also Figures S3 - S5 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure S1. Characterization of PDAC in KC mice. (A) Formalin-fixed paraffin-embedded (FFPE) sections from KC or control ( WT ) mice were stained with H&E or subjected to immunohistochemistry analysis using antibodies to Muc5AC and CK19. Representative pictures of normal pancreas, PanIN1, PanIN2, PanIN3, and full-blown PDAC are shown. Bar, 200 muM. (B, C) FFPE sections from KC or control mice were subjected to immunohistochemistry analysis using antibodies to CD31 (B) or insulin (C). Representative pictures of normal or cancerous tissues with remnant islets taken at different magnifications are shown. Bar, 400 muM (top), 200 muM (middle), and 100 muM (bottom). (D, E) FFPE sections from KC or control mice (n = 6) were immunostained with antibodies to insulin or glucagon and revealed by immunofluorescence. Insulin-positive (INS+) or glucagon-positive (GCG+) cells in all islets from six different sections were counted, and results are presented as percentage of INS+ or GCG+ cells relative to the total cell number in islets. Bar, 200 muM. Data are expressed as mean +- SEM. Statistical significance was estimated by unpaired t test. *** P < 0.001; ns, nonsignificant.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Document S1. Figures S1 and S2 and Tables S1 and S2