Anti-Sox9 antibody was purchased from Millipore (AB5535, Billerica, MA). Anti-β-catenin and glycogen synthase kinase 3β (GSK3β) antibodies were obtained from BD Transduction Laboratories (#610153 & #610201, San Jose, CA). Anti-Oct4, Sox2 and FZD7 antibodies were purchased from Abcam (ab19857, ab97959 & ab51049, Cambridge, UK). Anti-E-cadherin, vimentin and phospho-GSK3β (Ser9) antibodies were obtained from Cell Signaling Technology (#3195, #5741 & #9336, Beverly, MA, USA). Anti-β-actin antibody was purchased from Sigma-Aldrich (A5316, St. Louis, MO, USA). Anti-CD24 (ab30350, Abcam), E-cadherin (#610181, BD Transduction Laboratories) and vimentin antibodies (M0725, Dako, Glostrup, Denmark) were used for immunofluorescent staining. Anti-CK19 (ab52625, Abcam), anti-AFP (A0008, Dako) and anti-EpCAM (M0804, Dako) were used for immunohistochemistry.
M0804
Manufactured by Agilent Technologies
Sourced in United States
The M0804 is a compact and versatile lab equipment designed for general laboratory use. It features a digital display and intuitive controls for precise monitoring and adjustments. The core function of the M0804 is to provide reliable and accurate measurements for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab52625, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ab51049, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Anti β catenin, by BD (1 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
Ab19857, by Abcam (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Anti sox9 antibody, by Merck Group (1 mentions)
Gsk3β, by BD (1 mentions)
A0008, by Agilent Technologies (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
α sma, by Thermo Fisher Scientific (1 mentions)
Cxcl12, by Thermo Fisher Scientific (1 mentions)
Ab5535, by Merck Group (1 mentions)
Envision hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Protein block serum free solution, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
4 protocols using m0804
1
Immunodetection of Stem Cell Markers
2
Immunophenotyping of tumor-associated cells
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Approximately 2.5 × 104 of CAFs/DAFs were seeded on the slides and incubated overnight. PANC-1 cells were used in addition as control for epithelial cell adhesion molecule (EpCAM) staining. Cells were then fixed with 4% PFA and sequentially immunostained with antibodies directed against alpha-smooth muscle actin (α-SMA) (Invitrogen, 701457), KRT19 (Invitrogen, MA5-31977, Waltham, MA, USA), C-X-C motif chemokine 12 (CXCL12) (Invitrogen, PA5-114344), or EpCAM (Dako, M0804).
3
Immunohistochemical Analysis of Epithelial Markers
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IHC staining for EpCAM, CK19 and Sox9 were performed on formalin-fixed, paraffin-embedded sections using labeled horseradish peroxidase (HRP) method. After heat antigen retrieval with Tris- EDTA buffer (Sox9, CK19) or Protease K enzyme antigen retrieval (EpCAM), endogenous peroxidase activities were quenched by 3% H2O2. The sections were immersed in serum free-protein block solution (Dako) and incubated with anti-Sox9 (Millipore AB5535, MA at dilution 1:1000), anti-CK19 (ab52625, Abcam) at dilution 1:1000), and anti-EpCAM (M0804, Dako at dilution 1:100) at 4°C overnight. The sections were thoroughly washed and incubated with EnvisionTM HRP-conjugated secondary antibody (Dako). Positive signals were visualized using 3,3′-diaminobenzidine (Dako). Nuclei were counterstained with hematoxylin.
4
Quantifying DNA Damage in Organoids
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Organoids were expanded in µ-Slide 8-well ibiTreat chambers (Ibidi, Fitchburg, WI) and treated with 200 nM camptothecin (CPT) for 24 h. As a control, some wells were left untreated. Phosphorylation of the Ser-139 residue of the histone variant H2AX (γH2AX) was assessed by immunofluorescence staining following already published protocols (Mayorgas et al., 2021 (link)), with some modifications. After the fixation step, organoids were stored in PBS overnight at 4°C, and the next day the permeabilization and blocking steps were performed as indicated. Samples were then incubated with anti-γH2AX (#ab81299, 1:750, Abcam) and EpCAM (#M0804, 1:150, Dako) primary antibodies overnight at 4°C. The next day, samples were incubated with the secondary antibodies anti-rabbit Alexa Fluor® 594 and anti-mouse Alexa Fluor® 488 (ThermoFisher, Waltham, MA), both diluted 1:500. After DAPI nuclear staining, samples were overlaid with Ibidi Mounting medium (#50001, Ibidi, Fitchburg, WI) and stored at 4°C for subsequent fluorescent microscope observation in a Zeiss LSM 880 confocal laser scanning microscope (CCiTUB optical microscopy facility, Universitat de Barcelona, Spain). Positive nuclei (γH2AX–DAPI colocalization) were counted by using the CellCounter plug-in in ImageJ software. The ratio of γH2AX-positive nuclei versus the total number of nuclei per organoid was calculated.
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