Total proteins from ileal tissues or enteroids were extracted using tissue extraction reagent (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. The protein concentrations were quantified using a bicinchoninic acid protein assay kit (Thermofisher). Equal amounts of proteins (20 μg) were electrophoresed through a Express Plus PAGE gel (GenScript, M42015 C, M42010 C) and transferred to polyvinylidene fluoride membranes. Membranes were subsequently put into 5% (w/v) nonfat milk for 2 hours at room temperature, and then incubated with a primary antibody (Occludin, 1:500, Clone OC-3F10, Invitrogen, 33–1500; LGR5, 1:500, Boster Biological Technology, BM4244; PCNA, 1:500, Santa Cruz Biotechnology, sc-56; lysozyme, DAKO, 1:500, Glostrup, clone EC32117; GAPDH, 1:5000, CMCTAG, USA, AT0002; β-actin, 1:5000, CMCTAG, AT0001) overnight at 4°C. On the next day, membranes were incubated with second antibody for 1 hour at room temperature. The protein bands were visualized using an ECL kit (Perkin-Elmer Applied Biosystems) under GE system and quantified using Image J software.
Lysozyme
Manufactured by Agilent Technologies
Sourced in United States
Lysozyme is an enzyme that is commonly used in laboratory settings. It has the ability to break down the cell walls of certain bacteria, making it a useful tool for researchers working with microbial samples. Lysozyme's core function is to catalyze the hydrolysis of peptidoglycan, a key component of bacterial cell walls.
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Lab products found in correlation
β catenin, by BD (10 mentions)
Cleaved caspase 3, by Cell Signaling Technology (7 mentions)
Bromodeoxyuridine (brdu), by BD (7 mentions)
Olfm4, by Cell Signaling Technology (4 mentions)
Phospho histone h3, by Cell Signaling Technology (3 mentions)
C myc, by Santa Cruz Biotechnology (3 mentions)
Chromogranin a, by Abcam (3 mentions)
Shandon cytospin 4, by Thermo Fisher Scientific (3 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Mucin 2, by Santa Cruz Biotechnology (2 mentions)
E cadherin, by R&D Systems (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Triton x 100, by Merck Group (2 mentions)
E cadherin, by BD (2 mentions)
Sc1661, by Vector Laboratories (2 mentions)
Phospho rps6 ser235 236, by Cell Signaling Technology (2 mentions)
Phospho eef2thr56, by Novus Biologicals (2 mentions)
Nb100 92518, by Santa Cruz Biotechnology (2 mentions)
Vpp956, by Vector Laboratories (2 mentions)
30 protocols using lysozyme
1
Protein Expression Analysis in Ileal Tissues
2
Immunohistochemical and Western Blot Analysis
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β-Catenin (610154, BD); caspase-3 (9661L, Cell Signaling); cyclin D1 (2978S, Cell Signaling); lysozyme (A0099, Dako); Sox9 (AB5335, Millipore); Olfm4 (39141S, Cell Signaling); USP7 (Bethyl Laboratories); CD44 (MAB2137, Merck); and keratin 20 (13063S, Cell Signaling) were used in immunohistochemistry analysis. α-Actinin (sc-15335) and β-Catenin (610154, BD) were used for western blot analysis. DUB inhibitor VI P22077 (Calbiochem) was resuspended in DMSO at 10 mM (for organoid experiments) or 15 mg/mL (for mice experiments).
3
Immunohistochemical Analysis of Intestinal Tissues
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Intestines extracted from mice were processed for wholemount analysis or embedded in OCT, frozen, sectioned at 5–15 µm and fixed additionally for 10 min in 4% paraformaldehyde (PFA). Human colon samples were provided as paraffin-embedded sections (Oncology Division-Rambam Health Care Campus, Haifa), which were deparaffinized and rehydrated. Antigen retrieval was performed by boiling samples at 95 °C for 20 min. Sections were then blocked and incubated overnight at 4 °C with primary antibodies against: ARTS (Sigma; #SAB3500314 and #A447), β-catenin (Abcam; #ab32572), non-phosphorylated (active) β-Catenin (Cell Signaling; #8814), Ki67 (eBioscience; #14-5698-80), PCNA (Abcam; #ab18197), GFP (Abcam; #ab13970), lysozyme (Dako; #A0099), XIAP (BD; #610762), cleaved caspase-3 (Cell Signaling; #9661), REG4 (R&D Systems; #AF1379), Chromogranin A (Santa Cruz; #sc-376827), villin (Abcam; ab130751) and BrdU (Santa Cruz; #sc-32323). Sections were then incubated with secondary antibodies (Alexa Fluors 488, 546, or 633) for 1 h at room temperature. For TUNEL assays the Click-iT TUNEL Alexa Fluor 594 kit was used according to the manufacturer’s instructions. Sections were then mounted in Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) and visualized on a Zeiss LSM-880 confocal microscope.
4
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).
5
Multimodal Protein Analysis Protocol
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β‐catenin (610154, BD), caspase‐3 (9661L, Cell Signalling), Cyclin D1 (2978S, Cell Signalling), DVL2 (10B5) (sc‐8026, Santa Cruz), GAPDH (sc‐47724, Santa Cruz), Flag (F3165, SIGMA), HA (Y‐11) (sc‐7392, Santa Cruz), LGR4 (C‐12) (sc‐390630, Santa Cruz), Lysozyme (A0099, Dako), c‐Myc (9E10) (sc‐40, Santa Cruz), NEDD4 (sc‐25508, Santa Cruz), NEDD4L (4013S, Cell Signalling), SNAP (P9310S, NEB), Sox9 (AB5335, Millipore) and V5 (ab27671) were used in immunohistochemistry, immunoprecipitations or Western blot analysis.
6
Comprehensive Antibody Panel for Cellular Characterization
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Antibodies against the following proteins were purchased from commercial sources: afadin, chromogranin A, and DCAMKL (Dclk) (Abcam, Cambridge, UK); E-cadherin (R&D Systems, Minneapolis, MN, USA and BD Biosciences, San Jose, CA, USA); ZO-1 (Sanko-junyaku, Tokyo, Japan); Ki-67 (Novocastra Laboratories, Newcastle Upon Tyne, UK); lysozyme (DAKO, Glostrup, Denmark); cleaved caspase3 (Cell Signaling, Beverly, MA, USA); Rap1 (Millipore Corporation, Billerica, MA, USA); EphB3 (Abcam and R&D Systems); and EphB2 and ephrinB1 (R&D Systems). Alexa Fluor and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Millipore Corporation and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively.
7
Comprehensive Protein Analysis in Tissues
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Immunohistochemistry, immunofluorescence, and immunoblot analyses were performed using monoclonal antibodies against FGF21, CLCA3, cleaved caspase 3, BrdU, β-Klotho (Abcam, Cambridge, MA, USA), Lysozyme (DAKO, Carpinteria, CA, USA), E-Cadherin, phospho-Stat3, Stat3, phospho-Stat5, Stat5, phospho-Protein kinase B (Akt), Akt, Socs2, Socs3, Janus-activated kinase (Jak) 1, Bax, β-actin (Cell Signaling Technologies, Beverly, MA, USA), and β-Klotho (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
8
Histological Analysis of Tissue Samples
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5 μm thick paraffin sections were used for hematoxylin and eosin (H&E) staining, Alcian blue-staining, and immunohistochemical staining using standard procedures. Immunohistochemistry was performed using antibodies against Lysozyme (1:200, A0099, DAKO), E-Cadherin (1:200, #3195S, Cell Signaling), N-Cadherin (1:100, 610920, BD), Stat3 (1:200, #9139, Cell Signaling), beta-catenin (1:50, sc-7963, Santa Cruz), IL-4 (1:100, PAB16160, Abnova), iNOS (1:200, ab129372, Abcam), Arginase I (1:100, 610708, BD), and LSAB+System-HRP kit (K0679, DAKO). The slides were mounted and viewed on a Nikon Eclipse 80i microscope. Images were photographed with a SPOT RT3 CCD camera and SPOT Advanced software (Diagnostic Instruments, Sterling Heights, MI, USA).
9
Immunohistochemical and In Situ Analysis
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Standard immunohistochemistry techniques were used throughout this study. The following primary antibodies were used: BrdU (1/200, #347580, BD Biosciences), β-catenin (1/50 #610154, BD Biosciences), lysozyme (1/200, DAKO #A0099), RFP (1/200, Rockland #600-401-379). RNA in situ hybridisation (RNAscope) was performed according to the manufacturer’s protocol (ACD RNAscope 2.0 High Definition–Brown) for Lgr5 and Olfm4. BaseScope (also ACD) Apc EX14 #701641 (detects wild-type APC exon 14) and Apc E14E16 #703011 (detects floxed APC) were used according to the manufacturer’s instructions.
Staining for nuclear β-catenin and RNA in situ hybridisation was performed on tissue samples fixed at 4 °C for less than 24 h in 10% formalin prior to processing.
Staining for nuclear β-catenin and RNA in situ hybridisation was performed on tissue samples fixed at 4 °C for less than 24 h in 10% formalin prior to processing.
10
Comprehensive Immunohistochemical Analyses of Intestinal Tissues
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IHC and IF were performed as described previously (Dai et al., 2000 (link)), using formalin-fixed and paraffin-embedded jejunal sections and the following antibodies: human p16 (JC2, Jim Koh, Duke University), mouse p16 (M156, Santa Cruz Biotechnology, Santa Cruz, CA, USA) BrdU (IHC: Becton Dickinson, #347580; IF: Abgene), activated Caspase 3 (#9661, Cell Signaling Technology, Danvers, MA, USA), lysozyme (Dako, # EC.3.2.1.17), chromogranin A (Immunostar, #20085), PCNA (#2586, Cell Signaling), phospho-histone H3 (#9071, Cell Signaling), cyclin D1 (sc-753 (cross-reacts with cyclin D2, Santa Cruz), Cdk4 (#sc-260 (Santa Cruz), and γH2AX (#05-636, EMD Millipore, Billerica, MA, USA). Lgr5-lacZ expression was assayed by a modification of methods described previously (Barker et al., 2007 (link)). Freshly excised tissue was fixed in 2% neutral buffered formalin/0.2% glutaraldehyde/0.01% deoxycholate/0.2% NP40 for 30 min at RT, then incubated in X-gal substrate at RT overnight in the dark before embedding in paraffin. SAβgal staining was at pH 6.0. For p16 IB and Cdk4 IP, intestinal epithelial cells were isolated by treatment of excised tissue with EDTA (Weiser, 1973 (link)) and protein extraction with E1a lysis buffer (Harlow et al., 1985 (link)). For IB of γH2AX, mucosa was scraped off with a razor blade and chromatin extracts sheared by sonication.
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