Ab3649

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab3649 is a primary antibody product from Abcam. It is a polyclonal antibody that recognizes a specific target protein. This product is intended for use in various laboratory techniques.

Automatically generated - may contain errors

Lab products found in correlation

T7451, by Merck Group (3 mentions) Ab124762, by Abcam (3 mentions) Ab32575, by Abcam (2 mentions) Ab51054, by Abcam (2 mentions) Mca497rt, by Abcam (1 mentions) Immpact vector red alkaline phosphatase, by Vector Laboratories (1 mentions) H 3404, by Vector Laboratories (1 mentions) Immpact dab peroxidase substrate, by Vector Laboratories (1 mentions) Ab52625, by Abcam (1 mentions) 3 3 diaminobenzidine (dab), by Merck Group (1 mentions) Ab78486, by Abcam (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Odyssey clx imager, by LI COR (1 mentions) Irdye 680wc conjugated secondary antibody, by LI COR (1 mentions) Cleaved caspase 8, by Cell Signaling Technology (1 mentions) Ab79823, by Abcam (1 mentions) Ab13556, by Abcam (1 mentions) Eclipse ci l, by Nikon (1 mentions) Ab124804, by Abcam (1 mentions) Tcs sp8, by Leica (1 mentions)

24 protocols using ab3649

Comprehensive Immunohistochemical Analysis of Murine Tissue

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Tissues were fixed in 4% formalin, embedded in paraffin and sections were cut and mounted onto slides. Staining of mice tissue was performed with the following antibodies: anti-Muc5ac (Abcam #ab3649 1:100), anti-Cytokeratin19 (Abcam #ab52625 1:200), anti-ki67 (Abcam #15580 1:100), anti-Gkn1 (Thermofisher #PA547913 1:100), anti-Tgfbeta (Abcam #ab92486 1:100), anti-CD3 (Bio-Rad #MCA1477 1:250), anti-Ly6g (BD Pharmingen 551459 1:200), anti-CD3 (Abcam #ab21703 100ul), anti-Onecut2 (Abcam #ab28466 1:50), anti-F4/80 (Biorad #MCA497RT 1:200), anti-Tff1 (Abcam #ab190942 1:200), anti-Dcamkl1 (Abcam #ab37994 1:50), and anti-insulin (Dako, A0564, GP, 1:10) overnight at 4 °C.
ImmPACT DAB peroxidase substrate (Vector Laboratories #) and ImmPACT Vector red alkaline phosphatase (Vector Laboratories #VE-SK-5150) were used as substrates. Hematoxylin (Vector Laboratories #H-3404) was used as a counterstain.
We used N-Histofine kit, mousestain kit 414321F when staining with anti-Muc5ac (mouse antibody).

Schlesinger Y., Yosefov-Levi O., Kolodkin-Gal D., Granit R.Z., Peters L., Kalifa R., Xia L., Nasereddin A., Shiff I., Amran O., Nevo Y., Elgavish S., Atlan K., Zamir G, & Parnas O. (2020). Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells’ heterogeneity. Nature Communications, 11, 4516.

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Immunohistochemical Analysis of Ciliated and Goblet Cells

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Paraffin sections and insert membranes were investigated for the presence of ciliated cells and goblet cells using standard immunohistochemical procedures. Ciliated cells were determined by staining slides with a monoclonal mouse anti-acetylated α-tubulin antibody (Sigma-Aldrich T7451, St. Louis, MO, USA) and visualized with diaminobenzidine (DAB, Sigma) solution. In the same sections, goblet cells were subsequently visualized after staining with Alcian Blue. MUC5AC-positive cells were determined with a monoclonal mouse anti-MUC5AC antibody (Abcam, ab3649, Cambridge, UK) and visualized with 3-amino-9-ethylcarbazole (AEC, Sigma).

Song J., Heijink I.H., Kistemaker L.E., Reinders-Luinge M., Kooistra W., Noordhoek J.A., Gosens R., Brandsma C.A., Timens W., Hiemstra P.S., Rots M.G, & Hylkema M.N. (2017). Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD. Clinical Epigenetics, 9, 42.

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Immunohistochemical Analysis of Mucin and Aquaporin

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Tissue paraffin samples were cut into 3 μm sections. Thees sections were first deparaffinized in 1 mM ethylenediaminetetraacetic acid (EDTA) at 95 °C for 20 min, and then incubated with 3% hydrogen peroxide for 20 min. Subsequently, the sections were blocked using 10% goat serum for 90 min at room temperature. Primary antibodies against Muc5ac (Abcam, ab3649, 1:2000) or AQP5 (Abcam, ab78486, 1:2000) were applied, and the sections were left to incubate overnight at 4 °C. Subsequently, the sections were washed with PBS and incubated with a second antibody kit (Zhongshanjinqiao, Beijing, China) for 30 min at 37 °C. After rinsing in PBS, the sections were developed with 3,3′—diaminobenzidine (DAB). Finally, images were captured using a Leica light microscope. Immunohistochemical staining results were evaluated and quantified using ImageJ (Version 1.53).

Hao Y., Wang T., Hou Y., Wang X., Yin Y., Liu Y., Han N., Ma Y., Li Z., Wei Y., Feng W., Jia Z, & Qi H. (2023). Therapeutic potential of Lianhua Qingke in airway mucus hypersecretion of acute exacerbation of chronic obstructive pulmonary disease. Chinese Medicine, 18, 145.

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Molecular Profiling of Lung Inflammation

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Lung sections were prepared as previously described, then stained with mouse anti-MUC5AC antibody (#ab3649, Abcam), rabbit anti-α-SMA (#ab32575, Abcam), TLR4 (#ab13556, Abcam), rabbit anti-HMGB1 antibody (#ab79823, Abcam), rabbit anti-NLRP3 antibody (#214185, Abcam), rabbit anti-NLRC4 antibody (#A13117, ABclonal) and rabbit anti-Bcl-2 antibody (#A0208, ABclonal). After washing with PBS, slices were incubated with anti-rabbit secondary antibody for 30 minutes at room temperature. Signals were visualized with a DAB peroxidase substrate kit., and the localization of targeted molecules in the lung was assessed under a light microscope. Lung tissue samples were ground to fine powder under liquid nitrogen and lysates were subjected to SDS-PAGE and western blotting for the detection of the following antigens: TLR4 (#ab13556, Abcam), HMGB1 (#ab79823, Abcam), NLRP3 (#214185, Abcam), NLRC4 (#A13117, ABclonal), Bcl-2 (#A0208, ABclonal), procaspase-3 (#A0214, ABclonal), procaspase-8 (#108333, Abcam), cleaved caspase-3 (#9664s, Cell Signaling Technology) and cleaved caspase-8 (#9429, Cell Signaling Technology). After incubation with an IRDye^® 680WC-conjugated secondary antibody (LICOR Biosciences), immunoreactive bands were exposed to an Odyssey^® CLx Imager for image capture. Data analysis was performed with ImageJ Software.

Chen S., Deng Y., He Q., Chen Y., Wang D., Sun W., He Y., Zou Z., Liang Z., Chen R., Yao L, & Tao A. (2020). Toll-like Receptor 4 Deficiency Aggravates Airway Hyperresponsiveness and Inflammation by Impairing Neutrophil Apoptosis in a Toluene Diisocyanate-Induced Murine Asthma Model. Allergy, Asthma & Immunology Research, 12(4), 608-625.

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Uncovering Bile Duct MUC5AC-Sp1 Coexpression

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Fluorescent co-staining experiments were performed to verify the association between MUC5AC expression (cat. no. ab3649; Abcam) and Sp1 expression (cat. no. ab124804; Abcam) in bile duct tissue. IF was performed as the aforementioned IHC procedure. Tissues were incubated with fluorescent secondary antibody (1:100; cat. no. FITC-10835; ProteinTech Group, Inc.). Subsequently, sections were incubated with DAPI for 5 min to stain cell nuclei. The stained slides were visualized under fluorescence microscope (magnification, ×200 and ×400; Eclipse Ci-L; Nikon Corporation) and the images were analyzed using ImageJ 1.51K (National Institutes of Health). Quantitative analysis was completed based on the percentage of positive cells with MUC5AC and Sp1 staining (0–25%, low; 25–50%, medium; >50%, high).

Wu X., Yao C., Kong J., Tian Y., Fan Y., Zhang Z., Han J, & Wu S. (2020). Molecular mechanism underlying miR-130b-Sp1 transcriptional regulation in LPS-induced upregulation of MUC5AC in the bile duct epithelium. Molecular Medicine Reports, 23(2), 106.

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Characterization of Human Airway Organoids

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When models showed the cavity-like structure after 10–14 days of standard culture, the specific protein markers, including Trp63 (p63) for basal cells, acetylated α-tubulin (Ac-tub) for ciliated cells, mucin 5AC (MUC5AC) for goblet cells, and secretoglobin family 1A member 1 (SCGB1A1) for club cells, were used to stain different types of cells for characterizing the hAOs. In detail, the basement membrane matrix was depolymerized to harvest organoids from culture, and organoids were minimally washed with PBS and fixed in 4% paraformaldehyde for 10 min, blocked for 30 min, and incubated overnight with the primary antibodies p63 (1:200, ab124762, Abcam, United States), Ac-tub (1:200, T7451, Sigma, United States), MUC5AC (1:200, ab3649, Abcam, United States), and SCGB1A1 (1:200, NBP2-75705, Bio-Techne, United States). Then, hAOs were minimally washed and incubated with FITC-conjugated secondary antibody (1:200, Zenbio, China) or Cy3-conjugated secondary antibody (1:200, Zenbio, China) for 2–3 h. Finally, organoids were washed and incubated with DAPI stain for 10 min in the dark and were imaged by a confocal microscope (Leica, TCS SP8, Germany).

Jiang Y., Lu L., Du C., Li Y., Cheng W., Bi H., Li G., Zhuang M., Ren D., Wang H, & Ji X. (2023). Human airway organoids as 3D in vitro models for a toxicity assessment of emerging inhaled pollutants: Tire wear particles. Frontiers in Bioengineering and Biotechnology, 10, 1105710.

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Quantitative Analysis of MUC5AC in ALI

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MUC5AC staining was performed on the section of ALI membranes using mouse monoclonal MUC5AC antibody (Abcam; #ab3649) in 1:200 dilution as primary antibody and rabbit polyclonal anti-mouse IgG H&L (Abcam; #ab6728) in 1:1,000 dilution as secondary antibody. A minimum of five images were captured at random using Axio Imager M2 automated microscope (Carl Zeiss AG, Oberkochen, Germany) and processed using imageJ software, as described above.

Veerati P.C., Troy N.M., Reid A.T., Li N.F., Nichol K.S., Kaur P., Maltby S., Wark P.A., Knight D.A., Bosco A., Grainge C.L, & Bartlett N.W. (2020). Airway Epithelial Cell Immunity Is Delayed During Rhinovirus Infection in Asthma and COPD. Frontiers in Immunology, 11, 974.

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Immunocytochemical Staining of VCD-OOCs

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Immunocytochemical staining for α-smooth muscle actin (14-9760-82, Thermo Fisher Scientific, Waltham, MA) for hFM-DEC, cytokeratin(CK)-14 (ab51054: Abcam for ECTO, Cambridge, MA, USA), CK-18 (ab668; Abcam) and MUC5A (ab3649; Abcam) for ENDO, pan-cytokeratin (#4545, Cell Signaling Technology, Danvers, MA) for VEC, type I collagen (ab34710; Abcam) and vimentin (ab92547; Abcam) STROMA were performed after 48 h, as previously described.^{49 (link),57} Manufacturer’s instructions were used to calculate the staining dilutions to ensure uniform staining. After 48 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X, and blocked with 3% bovine serum albumin in 1× PBS before incubation with primary antibodies overnight at 4 °C. After washing with 1× PBS, the VCD-OOCs were incubated with Alexa Fluor 488–, 594, and 647–conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1:400 in 1× PBS for 2 h in the dark. The VCD-OOCs were washed with 1× PBS and treated with NucBlue^® Fixed ReadyProbes Reagent (R37606; Thermo Fisher Scientific) to stain the nucleus.

Tantengco O.A., Richardson L.S., Radnaa E., Kammala A.K., Kim S., Medina P.M., Han A, & Menon R. (2022). Modeling ascending Ureaplasma parvum infection through the female reproductive tract using vagina-cervix-decidua-organ-on-a-chip and feto-maternal interface-organ-on-a-chip. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(10), e22551.

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Quantifying Airway Mucin Expression

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BALF mucin electrophoresis and western blotting was performed using a slightly modified protocol as described previously (14 ). Briefly, BALF mucins were denatured with urea (6M). Equal volumes (50μl per well) of BALF samples were electrophoresed on 1% agarose gel (at 80V for 90 min) and vacuum-blotted onto nitrocellulose membranes with 4x sodium saline citrate buffer (SSC) for two hours at −50 mBar pressure. After transfer, nitrocellulose membrane was blocked with blocking buffer (LI-COR Biosciences, Lincoln, NE) for one hour at room temperature, followed by primary antibody incubation (overnight at 4°C) with rabbit polyclonal antibodies against MUC5B (UNC223, University of North Carolina, Chapel Hill, NC) and mouse monoclonal antibodies against MUC5AC (AB3649, Abcam, Cambridge, MA). After washing, the membrane was probed with IRDye 680RD Goat anti-rabbit and IRDye 800CW Goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE) secondary antibodies, each diluted to 1:5,000 in blocking buffer. After one hour of incubation at room temperature, the membrane was analyzed by Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE). The band intensity was measured using Image J software (NIH).

Lewis B.W., Vo T., Choudhary I., Kidder A., Bathula C., Ehre C., Wakamatsu N., Patial S, & Saini Y. (2020). Ablation of IL33 suppresses Th2 responses, but with sustained mucus obstruction, in the Scnn1b transgenic mouse model. Journal of immunology (Baltimore, Md. : 1950), 204(6), 1650-1660.

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Mucin Expression Profiling in Tissue Sections

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To evaluate mucin mRNA expression at the protein level, tissue segments were fixed for 24 h in 4% formaldehyde and subsequently embedded in paraffin. Five-micrometer cross-sections were deparaffinized, rehydrated, and used for immunohistochemical staining using target-specific primary antibodies and visualization with a secondary streptavidin–horseradish peroxidase antibody and 3-amino-9-ethylcarbazole (AEC) substrate to detect the expression and localization of MUC1 (AF6298, R&D systems, 1:500), MUC2 (NBP1-31231, Novus Biologicals, 1:3000), MUC4 (NBP1-52193, Novus Biologicals, 1:3000), MUC5AC (ab3649, Abcam, 1:5000), MUC6 (ab216017, Abcam, 1:50), and MUC13 (MABC209, Merck Millipore, 1:1000). The stained sections were analyzed by light microscopy (Olympus BX43) [30 (link)]. Distinct staining in more than 10% of the gastric cells was recorded as positive immunoreactivity for the relevant mucin. Tumors containing epithelial cells expressing only gastric or intestinal-type mucins were classified as having a gastric or intestinal mucin phenotype, respectively. Those containing cells expressing both gastric and intestinal type mucins were classified as having a mixed phenotype, whereas tumors with cells expressing neither gastric nor intestinal type mucins were classified as having a null mucin phenotype. The degree of immunostaining was evaluated by two independent observers.

Oosterlinck B., Ceuleers H., Arras W., De Man J.G., Geboes K., De Schepper H., Peeters M., Lebeer S., Skieceviciene J., Hold G.L., Kupcinskas J., Link A., De Winter B.Y, & Smet A. (2023). Mucin-microbiome signatures shape the tumor microenvironment in gastric cancer. Microbiome, 11, 86.

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