Whole-mount immunofluorescence assay was performed essentially as described previously (Zhang et al., 2012 (link); Xue et al., 2014 (link)). Primary antibodies for immunofluorescence were: mouse anti-GFP (1:200; sc-9996, Santa Cruz), rabbit anti-GFP (1:800; Ab290, Abcam), mouse anti-acetylated tubulin (1:400; T6793, Sigma), rabbit anti-aPKC (1:100; sc-216, Santa Cruz), rabbit anti-pH3 (1:200; #9701, Cell Signaling Technology), rabbit anti-active capase3 (1:200; BD 559565, BD Biosciences), mouse anti-GST (1:400; BE2075, EASYBIO), rabbit anti-p-MLC2 (Thr18/Ser19) (1:200; #3674, Cell Signaling Technology), and rabbit anti-RhoA (1:500; BS1782, Bioworld) antibodies. Secondary antibodies were Alexa Fluor 488- or 649-conjugated anti-mouse IgG (1:100; 115-545-003 and 115-605-003, Invitrogen) and Alexa Fluor 488- or 649-conjugated anti-rabbit IgG (1:100; A11008 and A27040, Invitrogen) antibodies. For imaging, the embryo region containing DFCs or KV was dissected, embedded in the mounting medium and were observed under a Zeiss LSM META confocal microscope. Confocal images of DFCs were acquired as a z-series with a step-size of 2 μm and those of KV as a z-series with a step-size of 1 μm.
Lsm meta confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM META confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a flexible and modular design that allows for customization to meet specific research needs. The core function of the LSM META is to provide high-resolution, high-contrast images of biological samples through optical sectioning and multi-channel detection capabilities.
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Lab products found in correlation
Zen 2009, by Zeiss (4 mentions)
Nanozoomer digital pathology slide scanner, by Hamamatsu Photonics (4 mentions)
Skor2, by Novus Biologicals (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
Calbindin, by Swant (2 mentions)
Vectashield dapi, by Vector Laboratories (2 mentions)
Ab290, by Abcam (1 mentions)
A27040, by Thermo Fisher Scientific (1 mentions)
Sc 216, by Santa Cruz Biotechnology (1 mentions)
Sc 9996, by Santa Cruz Biotechnology (1 mentions)
T6793, by Merck Group (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Alexa fluor 594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Hrp conjugated secondary antibody, by Vector Laboratories (1 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
13 protocols using lsm meta confocal microscope
1
Whole-mount Immunofluorescence Assay Protocol
2
Quantitative Nrf2 Immunocytochemistry
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After treatment, cells were fixed with 4 % paraformaldehyde for 20 min before blocking and permeabilizing in PBS containing 1 % BSA and 0.3 % triton X-100. Cells were subsequently incubated with antibody to Nrf2 (1:1000, Cell Signaling Technology (CST) #12721), MAP2 (1:1000; Chemicon, Merck Millipore), or GFAP (1:1000; Dako) overnight at 4 °C before further incubation with AlexaFluor-488, AlexaFluor-568, or AlexaFluor-647 secondary antibodies (1:250) for 1 h at room temperature. Before mounting onto microscope slides, cells were incubated with 0.5 μg/mL DAPI for 5 min. Cells were imaged with a Zeiss LSM Meta confocal microscope. Nuclear fluorescence intensity of Nrf2 was determined from approximately 500–700 cells per condition using ImageJ software.
3
tGFP-proAKAP4 Localization in HEK293T Cells
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HEK293T cells were plated on 18 mm cover slips and transfected using the calcium phosphate method with 1 μg tGFP-proAKAP4, tGFP-proAKAP4-T265A or GRASP5. 24 h after transfection, cells were serum starved (16 h, 0.1% FCS) and were pretreated with U0126 for 20 min and then PMA was added for various time points. Subsequently, the cells were washed with PBS, fixed in 4% paraformaldehyde (PFA) in PBS and then probed with DAPI. Cover slips were placed on glass slides with Vectashield mounting medium. Confocal fluorescent images were collected with a 63x magnification by Zeiss LSM-META confocal microscope.
4
Fluorescence Imaging of Nile Red and mVenus
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Fluorescence images were obtained using a Zeiss LSM Meta confocal microscope. Nile Red and mVenus were excited at 561 and 488 nm, imaged with HFT 405/488/561 and HFT 405/514/633 nm major beam splitters and fluorescence was detected at 571-593 and 507-539 nm, respectively.
5
Quantitative Analysis of Nrf2 and p65 Nuclear Translocation
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HUVECs were fixed in ice-cold methanol for 10 min, blocked with PBS/5% BSA and incubated at 4 °C overnight with rabbit anti-Nrf2 (1:200), rabbit anti-p65 (1:200) or negative control rabbit Ig fraction (DAKO), followed by washing and addition of either AlexaFluor goat anti-rabbit 488 (Invitrogen) or AlexaFluor donkey anti-rabbit 594 (Invitrogen) for 1 h at room temperature. Slides were washed, stained with Draq5 and mounted using Vectashield (Vector Laboratories, Peterborough, UK). Nuclear translocation was analysed using a Zeiss LSM META confocal microscope, with images obtained at 40× magnification from ten random fields of view. Fluorescence intensity of Nrf2 and p65 was quantified after correction for autofluoresence and defining threshold intensity from background fluorescence.
6
Fluorescent Immunohistochemistry Imaging
7
Immunolabeling of AKAP4 in Sperm
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Sperm were washed and smeared on polylysine precovered slides and then allowed to air-dry. Cells were washed with PBS, permeabilized with Triton X-100 0.5% buffered in PBS. Nonspecific binding was blocked with 3% BSA buffered in PBS. Cells were probed first with monoclonal anti-AKAP4 (1:75), washed three times with PBS, and then probed with a secondary anti-rabbit Alexa Fluor™ 488-labeled antibody. Slides were viewed with Zeiss LSM-META confocal microscope.
8
Immunostaining of Liver Proteins
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Immunostaining for GS, CPSI, and β-catenin was carried out as described previously9 with the following changes;
High temperature antigen retrieval was performed in EDTA buffer for CPSI, GS, and β-catenin. Following antigen retrieval sections were immuno-histochemically labelled for β-catenin or immunofluorescently labelled for CPSI and GS.
For immunohistochemistry sections were blocked in 10% normal goat serum (NGS) and 1% bovine serum albumin (BSA) in PBS. Antibodies were diluted in 1% NGS/1%BSA in PBS: anti-mouse β-catenin (1:50; Transduction Laboratories, San Diego, CA). Antibody detection was carried out using HRP conjugated secondary antibodies (1:200; Vector labs) and DAB (Vector Labs). All immunohistochemical staining was imaged using an Olympus microscope and CellP Olympus software.
For immunofluorescent staining with CPSI and GS sections were blocked in 5% BSA/PBS. Antibodies were diluted in 1% BSA/PBS CPSI (1:200, a generous gift from Wouter Lamers), GS (1:500, Transduction Labs), anti-mouse Alexa 488 (1:1000, Molecular Probes, Invitrogen) and anti-rabbit Alexa 594 (1:500, Molecular Probes, Invitrogen). Fluorescent staining was imaged using a Zeiss LSM Meta confocal microscope and LSM software.
High temperature antigen retrieval was performed in EDTA buffer for CPSI, GS, and β-catenin. Following antigen retrieval sections were immuno-histochemically labelled for β-catenin or immunofluorescently labelled for CPSI and GS.
For immunohistochemistry sections were blocked in 10% normal goat serum (NGS) and 1% bovine serum albumin (BSA) in PBS. Antibodies were diluted in 1% NGS/1%BSA in PBS: anti-mouse β-catenin (1:50; Transduction Laboratories, San Diego, CA). Antibody detection was carried out using HRP conjugated secondary antibodies (1:200; Vector labs) and DAB (Vector Labs). All immunohistochemical staining was imaged using an Olympus microscope and CellP Olympus software.
For immunofluorescent staining with CPSI and GS sections were blocked in 5% BSA/PBS. Antibodies were diluted in 1% BSA/PBS CPSI (1:200, a generous gift from Wouter Lamers), GS (1:500, Transduction Labs), anti-mouse Alexa 488 (1:1000, Molecular Probes, Invitrogen) and anti-rabbit Alexa 594 (1:500, Molecular Probes, Invitrogen). Fluorescent staining was imaged using a Zeiss LSM Meta confocal microscope and LSM software.
9
Immunostaining and Fluorescence Imaging of Amphibian Embryos
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For antibody staining embryos were embedded in fish gelatin as described previously (Chalmers et al., 2003) , except tadpole-stage (stage 30) embryos, which were incubated in 20% sucrose for 2 h, washed several times in PBS and then embedded in 15% fish gelatin for freezing. The embryos were cryosectioned and antibody stained as described (Chalmers et al., 2003) . The following antibodies were used: monoclonal anti-γ-tubulin produced in mouse clone GTU-88 (Sigma; T6557, 1 in 100), polyclonal rabbit anti-active caspase 3 (Abcam; ab13847, 1 in 100) rabbit polyclonal anti-histone H3 phospho S10 (Abcam; ab5168, 1 in 500). The following secondary antibodies were used: anti -mouse Alexa 568 (A-11004, Molecular Probes, Eugene, OR); anti-rabbit Alexa 568 (A-11011, Molecular Probes). All secondary antibodies were used at a 1 in 200 dilution. The nuclear stain, DAPI (Sigma, D9542, 1mg/mL) was used at 1 in 1000 dilution. Stained sections were mounted in Vectasheild (Vector Labroratories, Burlingame, CA) and imaged on a Zeiss LSM META confocal microscope (Thornwood, NY). GFP fluorescence was visualised directly without the use of antibody staining. All fluorescencent images were captured in the linear range of the confocal to allow quantification (see below).
10
Immunofluorescence Staining of β-Catenin and Flag
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Cells were cultured for 24 h on cover slips coated with poly-L-lysine (Sigma–Aldrich, St. Louis, MO, US). These cells were washed with PBS, fixed in methanol for 5 min at RT, and incubated with blocking buffer (1% BSA in PBS). These cells were then incubated with anti-β-catenin antibody (1:200, 8480, Cell Signaling) and anti-Flag M2 (1:200, F1804, Sigma-Aldrich) at 4 °C overnight. After washing with PBS three times, cells were incubated with secondary antibody (Alexa Fluor 594 chicken anti-rabbit, 1:200, Invitrogen) in blocking buffer at RT for 2 h. Cells were then washed with PBS three times and mounted using Fluorescent Mounting Medium with DAPI (GBI Labs, Mukilto, WA, USA). Images were acquired using a Zeiss LSM Meta confocal microscope (Carl Zeiss, Weimar, Germany) with an LSM Meta software. Image contrast and brightness were adjusted using an LSM image browser.
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