After 24-h treatments, EpiDerm-FT™ cultures were fixed in 10% formalin and later preserved by paraffin embedding. Sections (5 µm) were stained with hematoxylin and eosin (H&E) for evaluation of tissue morphology. Immunohistochemistry (IHC) was performed using anti-aquaporin-3 (AbCam; Cambridge, UK) and anti-keratin-10 (EMD Millipore; Billerica, MA). Secondary antibodies used were anti-rabbit-Alexa Fluor-488 and anti-mouse-Alexa Fluor-594 (Jackson Immuno Research Labs; West Grove, PA). Antibody specificity was confirmed by staining tissues with secondary antibodies only. Stained sections were viewed using an Olympus BX-41 microscope (Center Valley, PA) equipped with CCD camera (Hamamatsu Photonics K.K.; Shizuoka Pref., Japan) and using MetaMorph® software (Molecular Devices, Sunnyvale, CA).
Anti mouse alexa fluor 594
Manufactured by Jackson ImmunoResearch
Sourced in United States
Anti-mouse Alexa Fluor 594 is a secondary antibody conjugated with the red-fluorescent Alexa Fluor 594 dye. It is designed to detect and visualize primary antibodies raised against mouse antigens in various immunoassays, such as immunofluorescence and western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 anti rabbit, by Jackson ImmunoResearch (4 mentions)
Anti mouse alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ccd camera, by Hamamatsu Photonics (1 mentions)
Anti aquaporin 3, by Abcam (1 mentions)
Bx 41 microscope, by Hamamatsu Photonics (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Fabp4, by Abcam (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Mouse anti runx2, by Abcam (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Eclipse ti confocal microscope, by Nikon (1 mentions)
A 21271, by Thermo Fisher Scientific (1 mentions)
Sc7292x, by Santa Cruz Biotechnology (1 mentions)
Sc 6214, by Santa Cruz Biotechnology (1 mentions)
10 protocols using anti mouse alexa fluor 594
1
Evaluating Skin Barrier Proteins in EpiDerm-FT™
2
Immunofluorescence Analysis of Stem Cell Markers
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Cells were seeded on 96-well plates and treated with nano-siRNA complexes, fixed with 4% PFA for 20 min at room temperature, and permeabilized for 10 min using 0.1% Triton X-100 solution. Blocking was performed to suppress non-specific antibody binding by incubation with 10% donkey serum (Abcam, Cambridge, UK, ab7475) for 2 h. Subsequently, the cells were incubated with the primary antibodies against THY1 (1:200; Santa Cruz Biotechnology, CA, USA, SC-9163), CDH1 (1:200; Proteintech, Chicago, IL, USA, 20874-1-AP), PLZF (1:100; Invitrogen, USA, PA5-29213), Lin28 (1:200; Abcam, Cambridge, UK, ab46020), or SOX9 (1:200; Santa Cruz Biotechnology, CA, USA, SC-166505) at 4 °C overnight. Afterwards, samples were incubated with the corresponding secondary antibody anti-mouse-Alexa Fluor 594 (1:200; Jackson Immunoresearch, 715-585-151), anti-rabbit-Alexa Fluor 488 (1:300; Jackson Immunoresearch, 711-545-152), or anti-rabbit-Alexa Fluor 594 (1:300; Jackson Immunoresearch, 711-585-152) at room temperature for 1 h and counterstained with Hoechst 33342 (Beyotime Institute of Bio-technology, C1022). Negative controls were incubated with 10% donkey serum without the primary antibody. Finally, fluorescent images were acquired with an Olympus IX71 (Tokyo, Japan) inverted fluorescence microscope camera.
3
Adipocyte Differentiation Marker Analysis
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The fat miniatures were harvested, rinsed using PBS 3 times, and fixed with 4% paraformaldehyde (CellNest, Gyeonggi, Republic of Korea) for 30 min at room temperature. The samples were permeabilized with 0.3% Tween 20 (Sigma-Aldrich) in PBS for 1 h at room temperature and blocked with 0.3% Tween 20 and 3% bovine serum albumin (BSA; Millipore, MA, USA) in PBS for 1 h at room temperature. Next, primary antibodies were treated on samples for analysis against PPARγ (1:200; Santa Cruz Biotechnology), FABP4 (1:200; Abcam), YAP (1:200, Cell Signaling Technology), and actin (1:200, Abcam) at 4 °C overnight. The samples were then washed with PBS and treated with secondary antibodies [anti-mouse Alexa Fluor 488 and anti-mouse Alexa Fluor 594 (The Jackson Laboratory, Bar Harbor, ME, USA)] in PBS for 2 h at room temperature. Cell nuclei were counterstained with Hoechst 33342 (1:200; Thermo Fisher). Confocal imaging (LSM980; Zeiss, Oberkochen, Land Baden-Württemberg, Germany) and quantitative image analysis (NIH) were then performed.
4
Immunostaining of Stem Cell Markers
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After culturing in the O2 variant chip, we harvested cells, rinsed them three times with PBS, and fixed them with 4% paraformaldehyde (CellNest, Gyeonggi, Republic of Korea) for 30 min at RT. We permeabilized the samples with 0.3% Tween 20 (Sigma-Aldrich) in PBS for 1 hour at RT and then blocked them with 0.3% Tween 20 and 3% bovine serum albumin (Millipore, MA, USA) for 1 hour at RT. We treated the samples with primary antibodies overnight at 4°C for analysis of stemness [rabbit anti-SOX2 (1:100; Novus, CO) and mouse anti-OCT4 (1:100; Novus)], proliferation [rabbit anti-ERK (1:100; Cell Signaling Technology, MA) and rabbit anti-AKT (1:100; Cell Signaling Technology)], chondrogenesis [rabbit anti-aggrecan (1:100; Abcam) and mouse anti-Col2a1 (1:100; Millipore)], and osteogenesis [mouse anti-RUNX2 (1:100; Abcam) and mouse anti-COL1 (1:100; Millipore)]. We then washed the samples and treated them with secondary antibodies in PBS for 2 hours at RT. The secondary antibodies used included anti-mouse Alexa Fluor 488, anti-mouse Alexa Fluor 594, anti-rabbit conjugated to Alexa Fluor 488, and anti-rabbit conjugated to Alexa Fluor 594 (the Jackson Laboratory, Bar Harbor, ME, USA). We counterstained the nuclei with Hoechst 33342 (1:200), followed by confocal imaging (LSM980).
5
Immunohistochemical Analysis of Myenteric Neurons
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Preparations were similarly dissected for immunohistochemistry. They were stretched to a maximum length (nicardipine [3 μM] was added to the Krebs solution to facilitate maximal stretching), then fixed in Zamboni's fixative (4 °C for 48 h), cleared using dimethylsulfoxide (3 times for 10 min), then washed with phosphate buffered saline (PBS, 3 times for 10 min). Specimens were incubated in primary antibodies: goat antineuronal nitric oxide synthase (nNOS, 1 : 500, NB100‐858; Novus Biologicals, Littleton, CO, USA), mouse anti‐Hu (1 : 500, A21271; Molecular Probes, Eugene, OR, USA) for 48 h, rinsed in PBS and incubated in secondary antibodies: DyLight 405 donkey antigoat and antimouse Alexa Fluor 594 (1 : 200; Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 4 h. Tissues were viewed on an IX71 Olympus microscope (Olympus, Tokyo, Japan) or an Eclipse Ti confocal microscope (Nikon, Tokyo, Japan).
Images of four randomly selected ganglia, showing myenteric neurons immunoreactive (IR) for Hu and nNOS, were captured by confocal microscopy at 20× magnification. The number of neurons within each image was counted to calculate the total number of neurons/mm2. The soma‐dendritic area (μm2) of nNOS‐IR neurons was measured by tracing neuronal profiles, using Image J software (NIH, Bethesda, MD, USA). The average size of neurons was calculated from 10 cells per image.
Images of four randomly selected ganglia, showing myenteric neurons immunoreactive (IR) for Hu and nNOS, were captured by confocal microscopy at 20× magnification. The number of neurons within each image was counted to calculate the total number of neurons/mm2. The soma‐dendritic area (μm2) of nNOS‐IR neurons was measured by tracing neuronal profiles, using Image J software (NIH, Bethesda, MD, USA). The average size of neurons was calculated from 10 cells per image.
6
Immunofluorescence Assay for Lamin A/C
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Cells cultured on coverslips were fixed with 3% paraformaldehyde and permeabilized for 10 min in PBS/0.1% Triton X-100. Cells were blocked with 2% BSA in PBS/0.1% Tween (PBST-BSA) for 30 min and incubated with primary antibodies in PBST-BSA overnight at 4°C. Cells were incubated with secondary antibodies in PBST-BSA for 45 min and washed in PBST-BSA before mounting with DAPI (1:1000, D-9942, Sigma-Aldrich). Antibodies used were mouse anti-lamin A/C (1:1000, sc7292x, Santa Cruz Biotechnology), goat anti-pre-lamin A (1:100, sc6214, Santa Cruz Biotechnology), anti-mouse Alexa Fluor® 594 and anti-goat Alexa Fluor® 488 (both 1:1000, Jackson ImmunoResearch).
7
Immunofluorescence and Tumor Xenograft Analysis
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For immunofluorescence, frozen tissue sections or cell slides were permeabilized with 0.3% Triton X-100, blocked with 3% normal goat serum for 30 min, and incubated with anti-RUNX3 (Pathway Biological Technology), Ki-67 (Santa Cruz, CA), p21 (Sangon Biotech) or Bim (Sangon Biotech) overnight. The sections or slides were then incubated with anti-rabbit Alexa Fluor 488 (Jackson Immunoresearch, West Grove, PA, USA) or anti-mouse Alexa Fluor 594 (Jackson Immunoresearch), accordingly. DAPI was used for nuclear counterstaining. The samples were observed under a fluorescence microscope (Leica, Mannheim, Germany). Positive cells were counted blindly in 10 high power field (HPF)/section (×200).
Tumor xenograft experiment HGC-27 cells stably expressing empty vector or pcDNA-MT1JP were selected by G418 (Invitrogen, Carlsbad, CA). The indicated cells (100µl, 2×10 6 ) were inoculated subcutaneously into the dorsal right flank of each nude mouse (6 mice per group). Tumor volumes were calculated as 0.5×length×width 2 every 3 days. After 4 weeks, mice were sacrificed, and tumors were excised, weighed and immediately frozen at -80°C for future use.
Tumor xenograft experiment HGC-27 cells stably expressing empty vector or pcDNA-MT1JP were selected by G418 (Invitrogen, Carlsbad, CA). The indicated cells (100µl, 2×10 6 ) were inoculated subcutaneously into the dorsal right flank of each nude mouse (6 mice per group). Tumor volumes were calculated as 0.5×length×width 2 every 3 days. After 4 weeks, mice were sacrificed, and tumors were excised, weighed and immediately frozen at -80°C for future use.
8
Immunostaining of Paraffin-Embedded Tissue Sections
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For immunostaining on paraffin sections, dewaxed slides were washed three times with PBS followed a blocking step using 5% Donkey serum for 60 min. Anti-p63α (1:50, orb69390, Biorbyt), anti-CK15 (1:50, ab52816, abcam), anti-Ki67 (1:50, ab16667, abcam), and anti-caspase 3 (1:50, ab4051, abcam) were diluted in 2% donkey serum and incubated overnight at 4 C. After washing three times, Alexa Fluor 594 anti-mouse and Alexa Fluor 488 anti-rabbit (Jackson Immunoresearch) secondary antibodies were applied for 1 h at room temperature followed by three washing steps with 1x PBS. Sections were mounted using DAPI-Mowiol. Fluorescence was documented using a Leica Microscope (Leica DM 4000B, Leica, Wetzlar, Germany). As negative controls, matching Ig antibodies were used. N = 3 with three donor-different cell sheets per experiment and one hLESC donor per experiment.
9
Immunohistological Staining of Collagen
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For immunohistological staining, deparaffinized CS and γDCS sections were washed, blocked with 5% Donkey serum for 60 min and incubated over night with anti-collagen I (Sigma C2456), III (Sigma C7805), and IV (Sigma C1926) antibodies (dilution 1:50 in 2% donkey serum). Secondary antibodies (Alexa Fluor 594 anti-mouse (Jackson Immunoresearch), dilution 1:400 and Alexa Fluor 488 anti-rabbit (Jackson Immunoresearch), dilution 1:400) were applied for 1 h at room temperature followed by three intense washing steps and samples were mounted with DAPI-Mowiol. Fluorescence was documented using a Zeiss microscope (Zeiss AxioVertA1). N = 3 with two donor-different cell sheets per single experiment.
10
Immunocytochemistry of Myotube GFP-expression
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To investigate the long term GFP-expression among the various promotors in Myotubes, HSKM-Ab1167 myotubes were processed for immunocytochemistry (ICC) after 7 days of differentiation and C2C12 myotubes after a total of 11 days post application of the EPS protocol. Cells were washed in PBS and fixed for 10 min in ice-cold acetone. After blocking using 5% BSA in PBS for 1 h at RT and incubation with an Z-disk titin antibody (mouse-anti T12), kindly provided by Peter van der Ven and Dieter O. Fürst [33, 34 (link)] diluted 1:25 in 5% BSA/PBS at 4°C over night, an incubation with a corresponding secondary antibody (Alexa fluor 594 anti-mouse; 1:500; Jackson ImmunoResearch Europe Ltd., Ely, Cambridgeshire, UK) followed in 5% BSA/PBS at RT for 1 h. Between each antibody treatment cells were washed with PBS three times to remove previous antibody solutions. Imaging was performed on a fluorescence microscope (IX83 microscope, Olympus, Tokyo, Japan).
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