BrdU pulses in melanoma spheres were performed for 8 h (4 μg/ml). Spheres were plated on DMEM/Matrigel (1:40) (BD Biosciences) to allow attachment, fixed with 4% paraformaldehyde and processed as previously described.60 (link) For apoptosis, spheres were immunolabeled with a rabbit anti-cleaved Caspase3 (Cell Signaling Technologies, Danvers, MA, USA), followed by an anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody. For SOX2 immunofluorescence, spheres were immunolabeled with a rabbit anti-SOX2 antibody (AB5603; Millipore, Billerica, MA, USA) and with an anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody. Formalin-fixed paraffin-embedded sections were subjected to antigen retrieval (with citrate buffer pH 6.0), incubated with rabbit anti-SOX2 antibody (AB5603; Millipore) and rabbit anti-cleaved Caspase3 (Cell Signaling Technologies) and visualized using UltraVision Detection System (Lab Vision, Fremont, CA, USA) and diaminobenzidine (Dako, Carpinteria, CA, USA).
Ab5603
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
The AB5603 is a high-performance laboratory equipment designed for precise analytical measurements. It is a versatile instrument capable of performing a variety of tasks required in research and development laboratories. The core function of the AB5603 is to provide reliable and accurate data for scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (6 mentions)
Sc 5279, by Santa Cruz Biotechnology (4 mentions)
Ab5535, by Merck Group (4 mentions)
Novex ecl chemi substrate, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Ab10543, by Abcam (3 mentions)
Shandon immu mount, by Zeiss (3 mentions)
Mab5326, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa flour 488 anti rabbit, by Thermo Fisher Scientific (2 mentions)
Mab533, by Merck Group (2 mentions)
Anti e cadherin, by BD (2 mentions)
Mouse anti pax6, by Developmental Studies Hybridoma Bank (2 mentions)
Z1 apotome, by Zeiss (2 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor, by Abcam (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Mab4360, by Merck Group (2 mentions)
Mab4381, by Merck Group (2 mentions)
Ab15580, by Abcam (2 mentions)
52 protocols using ab5603
1
Melanoma Spheres: BrdU, Apoptosis, and SOX2
2
Immunostaining of Mouse ESCs and NPCs
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Mouse ESCs cultured on coverslips were washed twice with PBS and fixed with 4% paraformaldehyde for 15 mins at room temperature. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 mins and blocked in 0.1% Tween, 5% BSA in PBS for 30 min at room temperature. Primary antibody (anti-Rinf, 84546S, CST, 1:1000) and secondary antibody (Alexa Flour 488 -anti-rabbit, A21206, Life Technologies, 1:1000) incubations were carried out at room temperature for an hour. Nuclei were stained with DAPI (1:1000, 5ug/ml stock). Likewise, neural progenitors were stained with Nestin (MAB533, EMD Millipore Corp., 1:200) and Sox2 (AB5603, EMD Millipore Corp., 1:200) as described above. Similarly, ESCs cultured in TSC media were stained with anti-E-cadherin (610182, BD Bioscience, 1:200) and DAPI (1:1000, 5ug/ml stock). Microscopy was performed after final washes using Zeiss Axio Observer.A1 inverted microscope.
3
Immunohistochemical Evaluation of SOX2
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The TMAs were cut into 3 μm sections and dried on Flex IHC microscope slides (DakoCytomation, Glostrup, Denmark). Antigen retrieval was performed by heating the sections with Envision Flex Target Retrieval solution, high pH (Dako, Glostrup, Denmark). Staining was done at room temperature on an automatic staining workstation (Dako Autostainer Plus, Glostrup, Denmark) with anti-SOX2 rabbit polyclonal antibody (AB5603, Merck Millipore, Darmstadt, Germany) at 1:1000 dilution using the Dako EnVision Flex + Visualization System (Dako Autostainer, Glostrup, Denmark) and diaminobenzidine chromogen as substrate. Counterstaining with hematoxylin was the final step.
The IHC results were independently evaluated by two observers (JPR, and JMG-P), blinded to clinical data. SOX2 staining was evaluated as the percentage of cells with nuclear staining in the dysplastic epithelium or in the tumor tissue. SOX2 staining scores were classified as negative or positive staining on the basis of values below or above the median value of 10%. Since CSC-like subpopulations are usually limited to a very small percentage of cells, SOX2 staining in the dysplastic areas was also scored considering any positive nuclei.
The IHC results were independently evaluated by two observers (JPR, and JMG-P), blinded to clinical data. SOX2 staining was evaluated as the percentage of cells with nuclear staining in the dysplastic epithelium or in the tumor tissue. SOX2 staining scores were classified as negative or positive staining on the basis of values below or above the median value of 10%. Since CSC-like subpopulations are usually limited to a very small percentage of cells, SOX2 staining in the dysplastic areas was also scored considering any positive nuclei.
4
Quantifying Stem Cell Markers in BM Cells
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In-cell ELISA (62200, Invitrogen) was used to determine the relative protein levels in whole BM cells before and after coculture with MCs according to the manufacturer’s protocol with slight modification. It is an accurate and efficient method of analysis of protein levels in cells and is ideal since it can be performed on a 96-well format with multiple repeats and less cell number. Briefly, the BM cells were seeded and allowed to attach for 3–4 h following which they were grown subsequently alone or in coculture with LAD2 cells. All experiments were performed under reduced serum condition. After the stipulated time period, the wells were washed with ice cold PBS and then proceeded according to the kit protocol. Human SOX2 (AB5603, Merck Millipore) and human CD133 (Ab19898, Abcam) antibodies were used and the detected with a horseradish peroxidase conjugate. Cell number normalization was done with the whole-cell stain, Janus Green. Absorbance was detected using an ELISA plate reader.
5
Immunostaining of Mouse ESCs and NPCs
6
Immunofluorescence Staining of Tissue Sections
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Slides were rehydrated in PBS then blocked with 10% goat serum, 3% BSA, 0,4% Triton X-100 in PBS for one hour. Primary antibodies were incubated overnight diluted in the same solution at 4°C. The following primary antibodies were used in this study: chicken anti-GFP 1:1000 (1020 AVES), rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) 1:250 (Cell signaling #9101), rabbit anti-SOX2 1:500 (AB5603 Merck Millipore), mouse anti-TUJ1 1:500 (801202 Biolegend), rat anti-pHH3 1: 250 (S28, Abcam ab10543), rabbit anti-cleaved-caspase 3 1:500 (Asp175, CST 9661), mouse anti-PAX6 1:500 (Developmental Studies Hybridoma Bank), Phalloidin AlexaFluor 568 1:40 (ThermoFisher). The secondary antibodies used were: anti-chicken, anti-rabbit, anti-mouse or anti-rat with conjugated fluorochromes (Alexa Fluor 488, 568 or 647; ThermoFisher) at 1:500. Sections were incubated for one hour in the blocking solution containing Hoechst dye (1:1000). Slides were washed, mounted (Thermo Scientific Shandon Immu-Mount) and imaged with a Zeiss microscope Z1 Apotome or a confocal LSM 780.
7
Quantification of SOX2 Expression
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Cells were seeded into the 384-well plate at the density of 2000 cells/0.05 cm2. After 24 h, cells were fixed with 4% formaldehyde, permeabilized with 0.3% Triton X-100 and incubated in 1× PBS at pH 7.4 with 2% BSA (bovine serum albumin) for 40 min at room temperature to block non-specific antibody binding. Cells were incubated with primary rabbit anti-SOX2 antibody (dilution 1:100, AB5603, Merck KGaA, Darmstadt, Germany) for 1 h at room temperature. After 3 rinses with 1× PBS, pH 7.4, cells were incubated with secondary anti-rabbit Alexa 488 antibody (dilution 1:500; Merck KGaA, Darmstadt, Germany). Nuclei were counterstained with Hoechst (Dilution 6 μg/mL, Thermo Scientific™ Hoechst 33, 342 Solution (20 mM)). The fluorescent signal was measured at excitation/emission maxima of 496/519 using the CLARIOstar microplate reader (BMG LABTECH, Ortenberg, Germany). MARS Data Analysis Software (BMG LABTECH, Ortenberg, Germany) was used to analyze the obtained data. The data are presented as fold of change over control (RFU in the wells stained by normal rabbit IgG).
8
Immunocytochemistry: Protein Localization and Staining
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The slides were rehydrated in PBS and then blocked with 10% goat serum, 3% BSA, 0.4% Triton X-100 in PBS for one hour. The primary antibodies were incubated over-night diluted in the same solution at 4 °C. The following primary antibodies were used in this study: chicken anti-GFP 1:1000 (1020 AVES), rabbit anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody #9101 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-SOX2 1:500 (AB5603 Merck Millipore), mouse anti-Tuj1 1:500 (801202 Biolegend, California), rat anti-pH3 1: 250 (S28, Abcam ab10543), rabbit anti-caspase 3 1:500 (Asp175, CST 9661). The secondary antibodies used were as follows: anti-chicken, anti-rabbit, anti-mouse or anti-rat with fluorochromes (488, 568 or 647) at 1:500 (Alexa Fluor, Abcam, Cambridge). They were incubated for one hour in the blocking solution containing Hoechst (1:1000). F-actin staining was performed using phalloidin AlexaFluor568 (1:70) (Thermo Fisher, Waltham, MA, USA). The slides were washed, mounted (Thermo Scientific Shandon Immu-Mount) and imaged with a ZI Zeiss microscope equipped with an ApoTome or a confocal LSM 780.
9
Characterization of Pluripotent Stem Cells
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hESCs were passaged on Matrigel-coated plates, and cells were cultured in TeSR-E8 medium. Cells were fixed in 4% paraformaldehyde, and permeabilization and blocking were performed in 5% BSA and 1% Triton X-100 in PBS for 30 min. Cells were stained with anti-NANOG (Abcam, #AB80892), SOX2 (EMD Millipore, #AB5603), TRA-1-60 (EMD Millipore, #MAB4360), TRA-1-81 (EMD Millipore, #MAB4381), SSEA-4 (EMD Millipore, #MAB4304), or OCT4 (Santa Cruz Biotechnology, #SC-5279), all at 1:100. Secondary antibody was applied for 1 hr at room temperature. Nuclei were stained with DAPI (Life Technologies). Images were acquired using a Zeiss IX71 microscope and MetaMorph Advanced software.
10
Immunostaining of Pluripotency Markers in hESCs
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The hESC colonies were manually harvested under visual control, washed in 1× phosphate‐buffered saline (PBS), fixed in 4% formaldehyde for 15 minutes, washed three times in 1× PBS, permeabilized in 0.1% Triton‐X100 in PBS for 10 minutes/RT, blocked in 3% BSA in PBS for 1 hour. Then, cells were incubated with anti‐Sox2 (AB5603, Merck Millipore, Prague, Czech Republic, dilution 1:500) or anti‐Oct3/4 (sc‐5279, Santa Cruz Biotechnology, Heidelberg, Germany, dilution 1:100) primary antibodies overnight at 4°C. The cells were then washed three times in 1× PBS and incubated for 60 minutes room temperature with secondary antibody conjugated with AlexaFluor 568 (Sox2) or 488 (Oct3/4) and diluted 1:1,000 (Life Technologies/ThermoFisher Scientific, Prague, Czech Republic, A11004 or A11008, respectively), followed by additional wash in 1× PBS. Hoechst 33342 stain was added to the final concentration 5 µg/ml. Images were acquired using an automated microscope with ×10 objective (Image Xpress MicroXL, Molecular Devices).
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