Fixed ileum tissues were embedded in paraffin and 5-μm sections were prepared. The sections were incubated overnight in rabbit anti-MUC2 (NBP1-31231, Novus Biologicals, Littleton, CO, USA) at 4 °C. Then, the sections were washed with PBS three times and incubated in fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (bs-0295G, Bioss) for 1 h at room temperature in the dark. For visualization of cell nuclei, 4′,6-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories) was added. Images were captured under a fluorescent microscope (Nikon) at a magnification of 200×. Positively stained cells were counted in all villi for each tissue section.
Nbp1 31231
Manufactured by Novus Biologicals
Sourced in United States
NBP1-31231 is a lab equipment product offered by Novus Biologicals. It is designed for use in scientific research and laboratory settings. The core function of this product is to [Description not available].
Automatically generated - may contain errors
Lab products found in correlation
Af6298, by R&D Systems (2 mentions)
Nbp1 52193, by Novus Biologicals (2 mentions)
Mabc209, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole dapi h 1200, by Vector Laboratories (1 mentions)
Ab3649, by Abcam (1 mentions)
M3636, by Agilent Technologies (1 mentions)
Panoramic scan 150, by 3DHISTECH (1 mentions)
Casecenter 2.9 viewer, by 3DHISTECH (1 mentions)
Rm2255, by Leica (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 igg, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Clinisciences (1 mentions)
Ab15580, by Abcam (1 mentions)
Sp5 multi photon confocal microscope, by Leica (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Widefield microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
Ab97009, by Abcam (1 mentions)
A11008, by Abcam (1 mentions)
7 protocols using nbp1 31231
1
Quantifying Intestinal MUC2 Expression
2
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.
3
Immunohistochemical Analysis of Cell Markers
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The expression pattern of CDX2, MUC2 and methyl-CpG binding protein-2 (MeCP2) in the 10 cases were analyzed by immunohistochemistry. Paraffin-embedded tissues were cut and dewaxed through a series of graded alcohol. After antigen retrieval by microwave (Citrate, pH 6) for 10 min, endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min. The specimens were then incubated with 2% non-fat dry milk in phosphate-buffered saline (PBS) for 10 min and with primary antibodies against CDX2 (M3636, DAKO, Tokyo, Japan), MUC2 (NBP1-31231, Novus Biologicals, USA, CO) and MeCP2 (D4F3, Cell Signaling Technology, Inc., USA, MA) for 15 min. After three 10-min washes with PBS, the specimens were incubated with rabbit anti-mouse IgG antibody preabsorbed with non-immunized serum. Finally, the antigen-antibody complex was immunolocalized by the streptavidine-biotin peroxidase complex method.
4
Quantification of Colonic Muc2 Protein
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Mucin 2 (Muc2) proteins were labelled on formalin-fixed samples that were cut, using a microtome (Leica RM2255, Leica, Wetzlar, Germany), into 5-μm-thick sections and mounted on adhesive microscope slides (Adhesives slides KP printer, KliniPath, Duiven, The Netherlands). Immunostaining was performed using the Leica Bond RXm. Heat-induced antigen retrieval was done with Leica Bond™ Epitope Retrieval 1 (pH6) for 20 min. Rabbit polyclonal antibody against the MUC2 protein (NBP1-31231, Novus Biologicals, Littleton, CO, USA), diluted at 1/500, was revealed by a goat anti-rabbit Alexafluor 568 IgG (dilution 1/2000, Invitrogen, Waltham, MA, USA) with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, Invitrogen, Waltham, MA, USA) to counterstained nuclear. All the reagents were diluted with Bond Antibody Diluent (AR9352, Leica, Wetzlar, Germany). Slides were mounted using a fluorescent mounting medium (Fluoromount-G, Clinisciences, Nanterre, France), scanned with the Panoramic Scan 150 (3D Histech, Budapest, Hungary) and analyzed with the CaseCenter 2.9 viewer (3D Histech, Budapest, Hungary). Ten crypt (colon) or villus/crypt (ileum) units were analyzed in each sample, and the number of Muc2 cells was counted according to Fukuda et al. [17 (link)]. Tissue samples with non-well-oriented crypts were removed from the analysis.
5
Immunohistochemical Analysis of Intestinal Tissues
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Slides from Carnoy’s-fixed tissue samples were deparaffinized and subjected to antigen retrieval by steaming for 30 minutes in a sodium citrate buffer (10mM sodium citrate, pH 6.0, 0.05% Tween 20). Sections were blocked (HBSS, 2% goat serum, 2% FBS, 0.4% Triton X-100) and stained with antibodies against Muc2 (NBP1-31231; Novus Biologicals; diluted 1:500), Ki67 (ab15580; Abcam; diluted 1:100), or E-cadherin (CM1681; ECM Biosciences; diluted 1:200). Sections were then incubated with goat anti-rabbit Alexa Fluor 568 (Invitrogen) or goat anti-mouse Alexa Fluor 488 (Invitrogen) diluted 1:1,000-2,000. Coverslips were mounted using Vectashield containing DAPI (Vector Laboratories Inc.) and visualized using an upright Leica SP5 multi-photon/confocal microscope (Leica) or wide field microscope where indicated. For confocal microscopy, image z-stacks were taken at 40x magnification and images collected every 0.45μm; wide field images were taken at 20x magnification. Approximately 3-5 fields per sample were analyzed, with 2 samples per diet group from three independent experiments. The number of Ki67-positive nuclei present in the epithelial layer were counted in each field, the normalized to the number of crypts present in the section.
6
Validation of Rectal Organoid Markers
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To validate the organoids derived from rectum expression of rectal epithelial specific markers such as Muc2, ChgA, and CFTR were examined using immune-fluorescence analysis (supplement figure 1 ). In brief, rectal organoids were permeabilized with 0.1% Triton X-100 (Sigma #9002-93-1) in PBS for 30 min at room temperature, washed twice with PBS, and blocked with 5% normal goat serum (Life Technologies #50062Z) for 60 min. Organoids were incubated with primary antibodies (1:50 anti CFTR (Mouse IgM, Abcam #ab2784), 1:100 anti MUC2 (Rabbit IgG, Novus Biological #NBP1-31231) overnight at 4 °C followed by incubation with secondary antibodies (anti-IgG Rabbit Alexa Fluor 488 (Invitrogen #A11008) 8 μg/ml, 1:50 anti-IgM mouse Dylight 594 (Abcam #ab97009)) for 5 h at room temperature. For phospho-Histone H2A.X detection, organoids were stained with the Anti-phospho-Histone H2A.X (Ser139) antibody (2 μg/ml) (Mouse IgG1, Millipore #16-202A, clone JBW301, FITC conjugate).
7
Comprehensive Mucin Protein Profiling
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Five µm cross-sections were deparaffinized, rehydrated and used for immunohistochemical stainings using target specific primary antibodies and visualization with a secondary streptavidin-horseradish peroxidase-conjugated 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, 1:2000), MUC3 (NBP2-44434, Novus Biologicals, 1:100), MUC4 (NBP1-52193, Novus Biologicals, 1:3000) and MUC13 (MABC209, Merck Millipore, 1:1000). The stained sections were analyzed by light microscopy (Olympus BX43).
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