To examine the patterning of PMCs, embryos were immunolabeled using the 1D5 antibody (a kind gift from Dr. David McClay, Duke University) as previously described (McClay et al., 1983 ) with minor modifications. After microinjection, embryos were fixed overnight at 4 °C in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in filtered ASW. After washing for 10 min three times in PBS-Tween (PBS containing 0.05% Tween-20 [Bio-Rad, Hercules, CA]), embryos were blocked in 4% sheep serum in PBS-Tween for 1 h at room temperature. Embryos were incubated in 1D5 antibody at 1:100 dilution in 4% sheep serum in PBS-Tween at 4 °C for 36–48 h. They were then washed three times in PBS-Tween and incubated in Alexa 488 conjugated goat-anti-mouse antibody (ThermoFisher, Waltham, MA) at a 1:300 dilution in 4% sheep serum in PBS-Tween for 1 h at room temperature. After washing three times in PBS-Tween, they were counterstained with Hoechst 33342 (Lonza, Basel, Switzerland) at 1:1000 dilution in PBS-Tween for 5 min and washed three more times with PBS-Tween. Embryos were then imaged using Zen software with a Zeiss LSM 880 confocal microscope, or AxioVision software with a Zeiss Observer Z1 fluorescent microscope (Zeiss, White Plains, NY).
Observer z1 fluorescent microscope
Manufactured by Zeiss
Sourced in Germany
The Observer Z1 is a fluorescent microscope designed by ZEISS. It is a high-performance microscope system capable of capturing detailed images and videos of fluorescently labeled samples. The Observer Z1 utilizes advanced optics and illumination technologies to provide researchers with a versatile and reliable imaging platform for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Orca er camera, by Zeiss (2 mentions)
Lsm 710 confocal system, by Zeiss (2 mentions)
Lsm zen software, by Zeiss (2 mentions)
Improvision volocity software, by PerkinElmer (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti mouse cy2, by Jackson ImmunoResearch (2 mentions)
Anti p63 4a4 sc 8431, by Santa Cruz Biotechnology (2 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (2 mentions)
Qcm gelatin invadopodia assay kit, by Merck Group (2 mentions)
Pbs tween, by Bio-Rad (1 mentions)
Hoechst 33342, by Lonza (1 mentions)
Alexa 488 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Alexa fluor, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag antibody, by Merck Group (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Slowfade gold antifade, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
24 protocols using observer z1 fluorescent microscope
1
Immunolabeling of Pluteus Larvae for PMC Patterning
2
Immunofluorescence Analysis of Tumor Slices
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Tumour slices were fixed in 4% paraformaldehyde (Invitrogen, Cergy-Pontoise, France), washed with PBS, where after unspecific sites were blocked using PBS containing 10 mg ml−1 BSA for 1 h at room temperature (RT). Slices were then incubated with rat anti-human CD31 antibodies (550174BD Biosciences, San Jose, CA, USA), rabbit anti-human phospho-S6 ribosomal protein (pS6) (1/100), vimentin (1/100), or E-cadherin (1/100) primary antibodies (Cell Signaling, St Quentin en Yvelines, France) at RT for 1 h, followed by incubation with the secondary antibodies Alexa Fluor (1/200) (Molecular Probes, Life Technologies, Saint-Aubin, France) for 1 h at RT and in the dark. Nuclei were stained with 5 μg ml−1 DAPI. Image analysis was performed using Zeiss Observer Z1 fluorescent microscope (Zeiss, Oberkochen, Germany) and Image J software.
3
Visualizing HIV Envelope Protein Localization
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The HeLa-derived HLtat cells (2 × 105 cells/35 mm glass-bottomed plate) were transfected with 200 ng of HIV Env CE1-FLAG (plasmid 327H) and Env CE2-FLAG (330H). After 24 h, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton-X 100 in PBS, incubated with mouse anti-FLAG antibody (#F1804, Sigma, St. Louis, MO), followed by incubation with anti-mouse antibody conjugated with Alexa-Fluor 488 (Life Technologies, Carlsbad, CA, at 1:750 dilution each) as secondary antibodies. The nuclei were stained with DAPI (Life Technologies, Carlsbad, CA). Cells were visualized on a Zeiss Observer Z1 fluorescent microscope using Zeiss Axiovision software (Carl Zeiss Microimaging GmbH, Göttingen, Germany).
4
Immunofluorescence analysis of intracellular pathogens
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Infected macrophages were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at various times after the initial addition of the bacteria. Cells were then washed three times with PBS, permeabilized for 15 min with 0.01% Triton 100, and blocked for 1 h in PBS with 5% goat serum. Coverslips were incubated with primary antibodies directed against Rab5, EEA1, Rab7, LAMP1, calnexin, and giantin for 16 h at 4 C in PBS with 1% goat serum. Bacteria were detected using the primary monoclonal antibody Ed9 [9 (link)]. After washing three times in PBS with 1% goat serum, coverslips were incubated for 1 h with secondary antibodies in PBS with 5% goat serum. During this time, macrophage nucleic acids were stained with DAPI. After washing three times again with PBS and once with deionized water, SlowFade Gold antifade (Molecular Probes, Eugene, OR, USA) was added to the cover slips, which were mounted onto glass slides, and cells were observed on a Zeiss Observer Z1 fluorescent microscope (Zeiss, Thornwood, NY, USA). To determine the percentage of bacteria that co-localized with the different intracellular markers, a minimum of 100 ECV were counted for each marker. All assays were performed in triplicate.
5
Immunofluorescence Staining Protocol for Cell Analysis
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3.0 × 105 cells per well were seeded on glass coverslip inserted in 6-well plates and incubated overnight. The days after, the cells were fixed in 4% Paraformaldehyde (PFA) for 15 min. Then, the PFA solution was removed and the cells were rinsed twice with PBS 1X. After washing, the cells were incubated for 1 h with blocking solution (PBS 1X, 5% BSA, and 0.5% Triton X-100). Next, the cells were incubated overnight at 4 °C with primary antibodies (Table S4 ) diluted in blocking solution. The following day, after three washes with PBS 1X, the anti-mouse and anti-rabbit secondary antibodies conjugated with AlexaFluor 488 or AlexaFluor 594 (Invitrogen, Carlsbad, CA, USA) (diluted 1:1000) were added to the samples and incubated for 1 h at RT under agitation. The cells were washed with PBS 1X three times and cell nuclei were stained with 0.5 µg/mL Hoechst (Sigma Aldrich) (diluted 1:5000). Finally, the coverslips were mounted on glass slides and the images were acquired using a Zeiss Observer Z1 fluorescent microscope and ZEN 2012 software (Carl Zeiss Jena, Germany). About 30 to 35 cells were imaged for each condition. The list of primary antibodies used is in Table S4 .
6
Immunofluorescence Microscopy Protocol
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Immunofluorescence was performed using a Zeiss Observer Z1 fluorescent microscope (Zeiss) equipped with a Hamamatsu Orca-ER camera or a Zeiss confocal system LSM710 (Zeiss). Data acquisition and fiber-diameter measurements were performed using Improvision Volocity software (Perkin Elmer) or Zeiss LSM ZEN software (Zeiss).
7
Morphological Assessment of Hepatocytes
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To morphologically assess the cells, they were imaged on the designated days using a BX41 microscope (Olympus, Tokyo, Japan) or Zeiss Observer.Z1 fluorescent microscope (Zeiss, Dublin, CA). The previously described method was used to determine PHH attachment and percent confluency15 (link).
8
Immunofluorescence Microscopy Protocol
9
Immunohistochemical analysis of wound healing
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Formalin-fixed, paraffin-embedded skin biopsy samples of the wound were sectioned in 6-mm slices and stained manually. After deparaffinization in three successive 5-minute baths of xylene and rehydration in an ethanol gradient, antigen retrieval was achieved with EDTA buffer (1 mmol/L þ 0.05% Tween 20 adjusted to pH 8.0) for 20 minutes at boiling temperature and then washed for 15 minutes in water. Tissues were then permeabilized with 0.25% Triton X-100 in PBS for 30 minutes at room temperature and washed for 15 minutes in PBS. The sections were blocked with a blocking buffer (Life Technologies, Waltham, MA) for 1 hour at room temperature, washed, incubated at 4 C overnight with anti-CD206 (1/ 1000 dilution) antibody from Abcam (Toronto, Canada), and washed again before being incubated for 1 hour with donkey anti-rabbit Alexa Fluor 647 (1/100) secondary antibodies from Life Technologies. Slides were once more washed and finally counterstained using ProLong Gold Antifade Reagent with DAPI (Molecular Probes, Eugene, OR). Substitution of the primary antibodies with blocking buffer served as a negative control. A Zeiss Observer Z1 fluorescent microscope with the AxioVision rel4.8 program (Carl Zeiss, Oberkochen, Germany) was used for reading the slides. The examiner was blinded to the experimental conditions and took pictures at three random and constant areas for every section.
10
Orientation of Limb Ectoderm Cells
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Embryonic limbs were dissected in PBS, collected in 4% PFA and fixed for 12–16 h depending on the age, washed in PBS and cryoprotected. Cryostatic sections (12–15 μm thick) were collected on glass slides, blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at RT and incubated with the following primary antibodies, diluted from 1:250 to 1:50 in 1% BSA in PBS, ON at 4°C: anti-GM130 (BD), with anti-p63 (4A4 sc-8431, Santa Cruz), anti ZO-1 (InVitrogen) and with anti-E-cadherin (36/E 610182, BD). Sections were rinsed with PBS and incubated with secondary antibodies anti-mouse-Cy2 and anti-rabbit-Cy3 (Jackson ImmunoResearch) diluted 1:200, 1 h at RT, rinsed in PBS, counterstained with DAPI and examined with a Zeiss Observer-Z1 fluorescent microscope, equipped with the Apotome system.
For a semi-quantitative assessment of the orientation of the AER cells in whole-limbs, the results were visualized using the Rose2.0 software, showing the angle of each cell's longest axis as a unidirectional rosette graph divided into Bins of 15°. Each interval represents 25 cells per Bin. A minimum of N = 150 AER cells were counted for each of the two genotypes.
For a semi-quantitative assessment of the orientation of the AER cells in whole-limbs, the results were visualized using the Rose2.0 software, showing the angle of each cell's longest axis as a unidirectional rosette graph divided into Bins of 15°. Each interval represents 25 cells per Bin. A minimum of N = 150 AER cells were counted for each of the two genotypes.
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