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Mucin 2

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Mucin 2 is a high molecular weight glycoprotein that is a major component of mucus. It is responsible for the viscous and protective properties of mucus in various biological systems.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (3 mentions) Ly 6g ly 6c, by BioLegend (2 mentions) Claudin 3, by Abcam (2 mentions) P mlkl, by Abcam (2 mentions) F4 80, by BioLegend (2 mentions) Lysozyme, by Agilent Technologies (2 mentions) Ki67 antibody, by Abcam (2 mentions) Chromogranin a, by Santa Cruz Biotechnology (2 mentions) Lysozyme, by Diagnostic BioSystems (2 mentions) Villin, by Santa Cruz Biotechnology (2 mentions) Lsm800 confocal microscope, by Zeiss (2 mentions) Lysozyme, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Nacalai Tesque (1 mentions) Sars cov 2 nucleocapsid, by GeneTex (1 mentions) Sars spike glycoprotein, by Abcam (1 mentions) Villin 1, by Cell Signaling Technology (1 mentions) Chr a, by Santa Cruz Biotechnology (1 mentions) Slowfade, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Ultrasensitive s p kit, by Maixin Group (1 mentions)

Close spelling variants of this product

Mucin 2 rabbit polyclonal IgG Rabbit anti-mucin 2

12 protocols using mucin 2

1

Immunocytochemistry of SARS-CoV-2 Markers

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Immunocytochemistry was carried out as previously described.22 (link) Briefly, the cells were fixed, permeabilized, blocked, and incubated with primary antibodies against SARS-CoV-2 nucleocapsid (1:100; GeneTex, Irvine, CA, USA), SARS spike glycoprotein (1:100; Abcam, Cambridge, UK), villin-1 (1:100; Cell Signaling Technology, Danvers, MA, USA), lysozyme (1:100; Thermo Fisher Scientific), mucin-2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and chr-A (1:100; Santa Cruz Biotechnology) at 4 °C. The cells were then incubated with Alexa 488-conjugated (1:200; Thermo Fisher Scientific) or Alexa 594-conjugated (1:200; Thermo Fisher Scientific) secondary antibodies for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole DAPI (Nacalai Tesque, Kyoto, Japan). The cells were mounted in SlowFade (Thermo Fisher Scientific) and examined under a confocal laser-scanning microscope (Nikon A1; Nikon, Tokyo, Japan).
Yamada S., Noda T., Okabe K., Yanagida S., Nishida M, & Kanda Y. (2022). SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium. Journal of Pharmacological Sciences, 149(3), 139-146.
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2

Immunohistochemical Analysis of Cecum Tissue Sections

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Cecum tissue sections (5 µm) were deparaffinized and rehydrated using xylene and ethanol, respectively. Following antigen retrieval in citrate buffer (10 mM, pH = 6), samples were blocked in normal goat serum (5%) or donkey serum (5%). For immunohistochemistry, sections were stained with mucin 2 (Santa Cruz), claudin 3 (Abcam), p-MLKL (Abcam), Ly-6G/Ly-6c (BioLegend), and F4/80 (BioLegend) antibodies. Subsequently, specific staining was detected using the UltraSensitive S-P Kit and DAB Detection Kit (Maixin-Bio, China) according to the manufacturer’s directions. For immunofluorescence, tissue sections were stained with rabbit-anti-PCNA (Santa Cruz) and Alexa Fluor® 488-conjugated anti-rabbit IgG (Invitrogen). Epithelial cell apoptosis was analyzed by TUNEL staining using a commercial kit (KeyGEN Biotech). DAPI (1 µg/ml) was used to stain nuclei.
Yu S.X., Chen W., Liu Z.Z., Zhou F.H., Yan S.Q., Hu G.Q., Qin X.X., Zhang J., Ma K., Du C.T., Gu J.M., Deng X.M., Han W.Y, & Yang Y.J. (2018). Non-Hematopoietic MLKL Protects Against Salmonella Mucosal Infection by Enhancing Inflammasome Activation. Frontiers in Immunology, 9, 119.
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3

Organoid Immunohistochemistry Protocol

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Organoids were fixed in 4% paraformaldehyde overnight at 4 °C and resuspended in 2% low-melting agarose prior to paraffin embedding. 4-µm sections were cut and processed for immunohistochemistry analysis or Hematoxylin and Eosin (H&E) staining as previously reported.24 (link) The following primary antibodies were used: active caspase 3 (R&D systems, Abingdon, UK), Chromogranin A and Ki-67 (Abcam, Cambridge, UK), Mucin 2 (Santa Cruz Biotechnology, Heidelberg, Germany), and Lysozyme (Dako, Cambridge, UK).
Lorenzi F., Babaei-Jadidi R., Sheard J., Spencer-Dene B, & Nateri A.S. (2016). Fbxw7-associated drug resistance is reversed by induction of terminal differentiation in murine intestinal organoid culture. Molecular Therapy. Methods & Clinical Development, 3, 16024-.
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4

Immunohistochemical Analysis of Tumor Samples

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Freshly isolated tumors were fixed in 4% paraformaldehyde, dehydrated and mounted in paraffin using standard protocols. Standard immunohistochemical protocols were performed with the following antibodies (diluted 1:100): mouse anti-E-cadherin (BD Transduction Laboratories), rabbit anti-Ki67 (Abcam), Wnt7b (recognizing both Wnt7a and Wnt7b) (Novus Biological), Cleaved Caspase 3 (Cell Signaling), LEF1 (Cell Signaling), MUCIN2 (Santa Cruz). TUNEL staining was performed using In Situ Cell Death Detection Kit,TMR red (Roche, distributed by Sigma).
Secondary antibodies (dilution1:400) were anti-rabbit, anti-mouse, anti-chicken, antibodies conjugated with Alexa (A488, A594 or A647) from ThermoFisher Scientific. Images were taken using a Leica LSM 710 or Leica SP8 confocal microscope and processed using ImageJ (FIJI) and AdobePhotoshopCS6 software. Unless it is otherwise stated, tumor from minimally 6 independent animals (2-3 tumor per each animal) were checked and representative pictures are shown.
Degirmenci B., Dincer C., Demirel H.C., Berkova L., Moor A.E., Kahraman A., Hausmann G., Aguet M., Tuncbag N., Valenta T, & Basler K. (2021). Epithelial Wnt secretion drives the progression of inflammation-induced colon carcinoma in murine model. iScience, 24(12), 103369.
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5

Immunofluorescence Staining of Intestinal Organoids

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Culture medium was removed, and the organoids in Matrigel were washed with DPBS. Fixation was done with 4% Paraformaldehyde until the Matrigel was disintegrated. Permeabilization was performed with 0.1% Tween-20 and 0.2% Triton-X100 in PBS buffer, and then, 5% BSA in DPBS was used to reduce non-specific binding. Samples were covered with primary antibodies at 4 °C overnight. The next day, the samples were embedded with secondary antibodies at room temperature for 2 h. Antibodies used for the staining were as follows: Mucin 2 (Santa Cruz Biotechnology, Santa Cruz, CA), Ki67 antibody (Abcam, Cambridge, MA), Lgr5 (Abgent, San Diego, CA), E-cadherin (Santa Cruz Biotechnology), ChromograninA (Santa Cruz), β –catenin (Abcam), Lysozyme (Diagnostic Biosystems, Fremont, CA), α-SMA (Sigma-Aldrich), Vimentin (BD Biosciences), and Alexa Flour 488 conjugated anti-rabbit IgG and Alexa Flour 594 conjugated anti-mouse IgG (Life Technologies, Gaithersburg, MD).
Hahn S., Nam M.O., Noh J.H., Lee D.H., Han H.W., Kim D.H., Hahm K.B., Hong S.P., Yoo J.H, & Yoo J. (2017). Organoid-based epithelial to mesenchymal transition (OEMT) model: from an intestinal fibrosis perspective. Scientific Reports, 7, 2435.
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6

Intestinal Tissue Preparation and Immunostaining

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At times of sacrifice, mouse intestines were rinsed in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde overnight, rinsed in PBS, and either dehydrated for paraffin embedding or immersed in 30% sucrose for 4 hours at 4°C, embedded in OCT and frozen for cryosectioning. Antigen retrieval was achieved using citrate buffer, pH 6.0, with a pressure cooker (PickCell Laboratories). Foxl1 staining required the use of cryosections ,antigen retrieval, and amplification of signal using tyramide (TSA systems PerkinElmer). Antibodies used were: Ki67 (BD Pharmingen 1:500), Lysozyme (Dako 1:1000), Sox9 (Millipore 1:300), Mucin 2, HBEGF (Santa Cruz Biotechnology 1:50) Guinea Pig Foxl1 (1:1500), Goat GFP (Abcam 1:200), Mouse E-cadherin (BD transduction 1:250), Mouse β catenin (BD transduction 1:200), Rabbit EpCAM (Abcam 1:100), Rabbit αSMA (Abcam 1:100), Rabbit Myh11 (Abcam 1:100). Cy2-, Cy3- and Cy5-conjugated donkey secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. For immunohistochemistry, horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated at room temperature for 2 hours. VECTASTAIN ABC kit (Vector Laboratories) was then used to detect the signal, and the slides were washed with PBS. For signal development, the DAB substrate was used.
Aoki R., Shoshkes-Carmel M., Gao N., Shin S., May C.L., Golson M.L., Zahm A.M., Ray M., Wiser C.L., Wright C.V, & Kaestner K.H. (2015). Foxl1-Expressing Mesenchymal Cells Constitute the Intestinal Stem Cell Niche. Cellular and Molecular Gastroenterology and Hepatology, 2(2), 175-188.
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7

Organoid Immunocytochemistry Protocol

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Organoids immunocytochemistry had been developed previously [15 (link)]. Samples were thoroughly covered with primary antibodies at 4°C overnight. Antibodies used for the staining were as follows: mucin 2 (Santa Cruz Biotechnology, Santa Cruz, CA), Ki67 antibody (Abcam, Cambridge, MA), chromogranin A (Santa Cruz), lysozyme (Diagnostic BioSystems, Fremont, CA), mucin 5AC (Santa Cruz Biotechnology, Santa Cruz, CA), and villin (Santa Cruz Biotechnology, Santa Cruz, CA) The next day, the samples were embedded with secondary antibodies at room temperature for 2 h, and Alexa Fluor 594 conjugated anti-rabbit IgG and Alexa Fluor 594 conjugated anti-mouse IgG (Life Technologies, Gaithersburg, MD).
Jee J.H., Lee D.H., Ko J., Hahn S., Jeong S.Y., Kim H.K., Park E., Choi S.Y., Jeong S., Lee J.W., Cho H.J., Kwon M.S, & Yoo J. (2019). Development of Collagen-Based 3D Matrix for Gastrointestinal Tract-Derived Organoid Culture. Stem Cells International, 2019, 8472712.
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8

Murine Intestinal Organoid Staining

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Murine intestines were prepared as described previously5 (link). In situ hybridisation (ISH) assay was carried out as described previously5 (link). Organoids were immuno-stained either as whole-mount samples or as paraffin sections. Samples for IHC were processed as outlined before5 (link),24 and the following primary antibodies were used: ZEB2/SIP1 (H260; Santa-Cruz, or from Dr. Tulchinsky), E-cadherin (610181; BD), Vimentin (RV202; Santa-Cruz), α-SMA (ab5694; Abcam), Ki-67 (M7249; Dako) and Mucin2 (H300; Santa-Cruz). For IF, samples were exposed to goat anti-rabbit antibodies conjugated to Alexa Fluor594 (A11037; Invitrogen) and/or rabbit anti-mouse antibodies conjugated to Alexa Fluor488 (A11059; Invitrogen). Tetramethylrhodamine-B isothiocyanate (TRITC)-conjugated phalloidin (P1951; Sigma) was used to label actin filaments according to the manufacturer’s instruction.
Li N., Babaei-Jadidi R., Lorenzi F., Spencer-Dene B., Clarke P., Domingo E., Tulchinsky E., Vries R.G., Kerr D., Pan Y., He Y., Bates D.O., Tomlinson I., Clevers H, & Nateri A.S. (2019). An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance. Oncogenesis, 8(3), 13.
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9

Comprehensive Histological and Immunohistochemical Analysis of Tissue Samples

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For histology, tissue samples of muscle, duodenum and cecum were fixed in 4% buffered formalin (Macklin, #P804536) solution and sections were stained with H&E to examine morphologic changes. For immunohistochemistry, tissue sections were stained with Ly-6G/Ly-6c (BioLegend, #108419), F4/80 (BioLegend, #123119), mucin 2 (Santa Cruz, #sc-515032), claudin 3 (Abcam, #A2946), and p-MLKL (Abcam, #ab196436) antibodies. For immunofluorescence, sections were stained with histone primary antibody (ABclonal, #A2348) and Alexa Fluor® 488-conjugated anti-rabbit IgG secondary antibody (Abcam, #ab150077). Meanwhile, apoptotic cell death in tissues were analyzed by TUNEL staining using a commercial kit (KeyGEN BioTECH, #KGA7061) following the manufacturer’s instructions. DAPI (2 μg/mL) was used to stain nuclei. After sequential excitation, images were acquired utilizing a Laser Co-focus light microscopy (Nikon). For mucins secretion and glycosylation patterns assays, duodenal and cecal tissues were collected and stained with alcian blue as well as periodic acid–Schiff’s reagent (AB-PAS staining, Solarbio, #1285) according to the manufacturer’s manual.
Liu Y., Xing L.H., Li F.X., Wang N., Ma Y.Z., Li J.W., Wu Y.J., Liang J., Lei Y.X., Wang X.Y., Meng F.H., Yang Y.J., Li G.P., Wang X, & Yu S.X. (2022). Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis. iScience, 25(10), 105121.
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10

Immunofluorescence Staining of Organoids and Neurons

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Whole mIOs were fixed overnight in fresh 4% paraformaldehyde. Fixed organoids were unmasked in PBS through shaking at room temperature and were blocked and permeabilized with 5% BSA and 0.1% Triton X-100 in PBS. The primary antibodies that were used were Villin (Cat. sc-58897, Santa Cruz Biotechnology Inc., Dallas, TX, USA), Mucin 2 (Cat. sc-15334, Santa Cruz Biotechnology Inc., Dallas, TX, USA), Chromogranin A (Cat. ab15160, Abcam, Cambridge, UK), and Lysozyme (Cat. ab108508, Abcam, Cambridge, UK). Human neuronal cells containing dopaminergic neurons were fixed with 80% Methanol for 10 min. After blocking with 5% BSA, the primary antibodies that were used were MAP2 (Cat. ab5392, Abcam, Cambridge, UK) and TH (Cat. Ab152, Millipore Sigma, Burlington, MA, USA). The primary antibodies were bound overnight at 4 °C. The secondary antibodies that were used were donkey anti-mouse and anti-rabbit Alexa Fluor 488 and 594 (Invitrogen, Carlsbad, CA, USA). The secondary antibodies were bound for 1 h at room temperature. DNA was stained with Hoechst 33342 (Cat. H3570, Thermo Fisher Scientific, Waltham, MA, USA). The images of the crypt organoids and human neuronal cells containing dopaminergic neurons were acquired using an LSM 800 confocal microscope with Zeiss software.
Sim H., Lee J.E., Yoo H.M., Cho S., Lee H., Baek A., Kim J., Seo H., Kweon M.N., Kim H.G., Jeon Y.J., Son M.Y, & Kim J. (2020). Iroquois Homeobox Protein 2 Identified as a Potential Biomarker for Parkinson’s Disease. International Journal of Molecular Sciences, 21(10), 3455.
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