Mice were anesthetized by intraperitoneal injection of pentobarbital (200 mg/kg) for perfusion with ice-cold Dulbecco’s PBS with Heparin. Following perfusion, the skull cap was carefully removed and fixed overnight in 4% paraformaldehyde. Intact meninges were then peeled off and stored as floating sections in PBS until immunohistochemistry. For LYVE1 staining, meninges were washed in PBS-Triton X-100 (0.5%) and blocked in PBS-T with 0.5% BSA at room temperature. Sections were incubated with LYVE1-e660 (ThermoFisher) at 1:200 overnight at 40 C. Sections were washed in PBS-T prior to mounting on slides in Prolong Gold antifade reagent with DAPI (ThermoFisher) mounting medium. Slides were imaged on Zeiss Axio Imager Z2 fluorescence microscope and Cytation 5 imaging reader (Biotek Imaging, Inc.). Images were processed on ZEN software suite (Carl Zeiss, Inc.) and Cytation 5 imaging reader.
Zen software suite
Manufactured by Zeiss
Sourced in Germany
The ZEN software suite is a comprehensive imaging and analysis platform developed by Zeiss. It provides a user-friendly interface for managing and processing images acquired from Zeiss microscopes and imaging systems. The software offers a range of tools and features for image acquisition, visualization, and analysis, enabling researchers and scientists to effectively utilize Zeiss imaging technology.
Automatically generated - may contain errors
Lab products found in correlation
Imaris software, by Oxford Instruments (2 mentions)
Fluoview fv1000, by Olympus (2 mentions)
Environment chamber, by Pecon (2 mentions)
Aox antibodies, by Eurogentec (2 mentions)
Lsm 880 laser confocal with airyscan, by Zeiss (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Slowfade gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Labtek 2 4 chamber slides, by Thermo Fisher Scientific (1 mentions)
Click it edu labeling kit for microscopy, by Thermo Fisher Scientific (1 mentions)
Click it kit with alexafluor 488, by Jackson ImmunoResearch (1 mentions)
M58 monoclonal antibody, by Jackson ImmunoResearch (1 mentions)
Alexafluor 594 conjugated secondary anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Lsm 700 laser confocal microscope, by Zeiss (1 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
Vibratome vt1000, by Leica (1 mentions)
Lsm 800 confocal laser scanning microscope, by Zeiss (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
10 protocols using zen software suite
1
Meningeal Lymphatic Vessel Imaging in Mice
2
Multicolor Confocal Microscopy of Cell Markers
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Microscopic observations were obtained by using an Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus America, Melville, NY.), with a PlanApo 60x oil immersion lens, numerical aperture 1.4. CD45-FITC was excited with a 473 nm diode laser (15mW), with an emission setting of 520 nm. The cKit PE was excited with a 559 nm diode laser (18 mW) with an emission setting of 567 nm. CD144 AlexaFluor647 was excited with a 635nm laser (20 mW) with an emission setting of668 nm. The Imaris software (Bitplane AG) was used for the 3D rendering. For the time-lapse experiments, cells were evaluated in a PECON environment chamber (PeCon GmbH) that maintains a temperature of 37 °C and a CO2 concentration of 5%. Time lapse images were taken every 10 min during a period up to 24h. Zeiss Apotome microscope (Zeiss Canada, Toronto, ON) and ZEN software suite (Carl Zeiss, version 2012) were used for the image acquisition. CD45 APC was excited with a Mercury light. The excitation setting was 592-650nm. The emission setting was a 665-735 nm. A transmitted light image was made at the same time as the fluorescent image. The cultures were imaged with a 20x Plan-Apochromat lens, N.A. 0.8.
3
Quantifying Single and Double Positive Cells
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To determine if cells were single and double positive for a given marker, image analysis was performed in the Zen software suite (Carl Zeiss; Oberkochen, Germany). DAPI-positive cells that expressed at least one marker were identified and marked. The signal intensity of the FITC and PE channels was quantified after autofluorescence correction and if a cell demonstrated a signal intensity in FITC or PE of greater than 95% of the background level (based on an area intensity histogram) it was called positive for that marker. Clear areas of intense autofluorescence (e.g., blood cells) and/or process/staining artefacts were excluded from the analysis.
All statistics were performed in GraphPad Prism 6.05 (La Jolla, CA, USA). A two-way analysis of variance was used to determine if significant differences existed between the mean numbers of cell profiles for each cell type. Comparisons were made both within a joint, as well as between osteoarthritic and normal joints. A Bonferroni analysis was used for all post-hoc analyses. Alpha was set at 0.05.
All statistics were performed in GraphPad Prism 6.05 (La Jolla, CA, USA). A two-way analysis of variance was used to determine if significant differences existed between the mean numbers of cell profiles for each cell type. Comparisons were made both within a joint, as well as between osteoarthritic and normal joints. A Bonferroni analysis was used for all post-hoc analyses. Alpha was set at 0.05.
4
Phospho-ATM Localization in MDSCs
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MDSCs were seeded unto sterile No.1 (13 mm diameter) glass coverslip-inserts (VWR) at 1 × 104 per well of a 24-well plate and maintained overnight at routine culture or experimental conditions. Cells were stained with anti-CD33 PE, washed in ice-cold PBS, fixed in 2% Paraformaldehyde for 20 min at RTP followed by permeabilisation in 0.1% Triton X for 10 min. After permeabilisation coverslips were blocked in a blocking buffer consisting of 5% heat inactivated goat serum (HiNGS) in PBS for 1 h at RTP. Coverslips were then incubated for 1 h with an eFluor 660 conjugated anti human Phospho-ATM (Ser1981) antibody (Clone 10H11.E12 3G5, eBiosciences) diluted 1:100 in 5% HiNGS/PBS. The coverslips were washed air dried, then mounted in SlowFade gold antifade mountant with DAPI (ThermoFisher Scientific). Cells were examined by fluorescence microscopy using a Zeiss LSM 780 fluorescence confocal microscope and images acquired using ZEN software suite (Carl Zeiss Microscopy).
5
Adenovirus Infection of Arrested WI-38 Cells
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As described [2 (link)], WI-38 cells were grown until 100% confluent on LabTek II 4-chamber slides (Thermo-Fisher, Waltham, MA, USA). After becoming fully confluent, cells were incubated for a further 72 h to achieve growth arrest. Infections were carried out as described above with a multiplicity of infection (MOI) of 100 for dl309 [16 (link)], or dl311 [16 (link),17 (link)], or dl1116 [18 (link)], or dl1132 [19 (link)], or dl1133 [5 (link)], dl1134 [5 (link)], or dl1135 [5 (link)], or dl1136 [5 (link)]. One hour prior to fixation, cells were pulsed with 5-ethynyl-2´-deoxyuridine (EdU) for 1 h as per manufacturer’s specifications using the Click-It EdU labeling kit for microscopy (Life Technologies, Carlsbad, CA, USA). After EdU labeling, cells were fixed in 3.7% formaldehyde, stained for EdU using the Click-It kit with AlexaFluor 488 and labelled for E1A using M58 monoclonal antibody and AlexaFluor 594 conjugated secondary anti-mouse antibody (Jackson ImmunoResearch). Cells were visualized using LSM700 laser confocal microscope (Carl Zeiss AG, Oberkochen, Germany) and ZEN software suite (Carl Zeiss AG, Oberkochen, Germany).
6
Multicolor Confocal Microscopy of Cell Markers
7
Microdroplet Fabrication and Fluorescence Imaging
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To prepare microdroplets, 1 mL reaction solution containing 100 mM PBS buffer (pH 6.5), 0.33 g L−1 GCN, and 1 g L−1 RITC-labeled SsCR were mixed with 10 mL n-nonane in a microtube, placed in a bath sonicator (Kunshan KQ-300DE), and was sonicated for 2 min at room temperature. The prepared microdroplets were cast onto a glass slide and imaged using a confocal microscope (LSM 710 Meta, Carl Zeiss, GmbH, Germany). The fluorescence signals were recorded with excitation laser beams (405 nm for GCN and 488 nm for RITC-labeled SsCR) and their preprogrammed emission windows. The total frame time for a 512 × 512 pixel image was 0.8 s. The acquired images and fluorescence intensities were processed with the ZEN software suite (Carl Zeiss GmbH, Germany).
8
Mitochondrial Localization of AOX Protein
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Naegleria gruberi was seeded onto sterile poly‐L‐lysine treated glass coverslips in a 6‐well plate and incubated overnight at 25°C. The following day, the cells were treated with 250 nM MitoTracker Red for 30 min. The media was then aspirated, and cells were washed with 1× PBS, followed by fixation using 2% formaldehyde for 20 min. After fixation, 2% formaldehyde was removed and cells were permeabilized with 0.1% triton‐X 100 for 10 min. After permeabilization, cells were washed three times with 1× PBS and blocked using 3% bovine serum albumin in PBS for 1 h. Primary antibody staining was carried out at room temperature for 1 h with custom‐made AOX antibodies (Eurogentec; Peptide: 1911009, Rabbit 237), diluted to 1:1000. Secondary antibody staining was carried out for 1 h in the dark using anti‐Rabbit‐IgG‐Alexa 488. Slides were washed and mounted using Prolong Gold Antifade with DAPI. Laser Confocal Microscopy was carried out using the LSM 880 Laser Confocal with Airyscan by Zeiss. Laser sets used were 405, 488, and 594, with airyscan detector plate imaging for high resolution. Images were captured and analyzed using Zen software suite by Zeiss.
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Naegleria gruberi was seeded onto sterile poly‐L‐lysine treated glass coverslips in a 6‐well plate and incubated overnight at 25°C. The following day, the cells were treated with 250 nM MitoTracker Red for 30 min. The media was then aspirated, and cells were washed with 1× PBS, followed by fixation using 2% formaldehyde for 20 min. After fixation, 2% formaldehyde was removed and cells were permeabilized with 0.1% triton‐X 100 for 10 min. After permeabilization, cells were washed three times with 1× PBS and blocked using 3% bovine serum albumin in PBS for 1 h. Primary antibody staining was carried out at room temperature for 1 h with custom‐made AOX antibodies (Eurogentec; Peptide: 1911009, Rabbit 237), diluted to 1:1000. Secondary antibody staining was carried out for 1 h in the dark using anti‐Rabbit‐IgG‐Alexa 488. Slides were washed and mounted using Prolong Gold Antifade with DAPI. Laser Confocal Microscopy was carried out using the LSM 880 Laser Confocal with Airyscan by Zeiss. Laser sets used were 405, 488, and 594, with airyscan detector plate imaging for high resolution. Images were captured and analyzed using Zen software suite by Zeiss.
9
Tissue Preparation for Electrophysiology and Histology
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For electrophysiology experiments, the tissue block containing the injection site was stored overnight in 10% neutral-buffered formalin at 4°C. After, it was transferred into 0.2% PBS Azide at 4°C until sectioning. The tissue block was mounted onto a stage and sectioned in 0.1M PBS at a thickness of 100μm using a VT-1000 vibratome (Leica). Slices were mounted onto microscope slides using DAPI Fluoromount-G (Southern Biotech #0100–20) and coverslipped before confocal imaging.
Following all behavioral experiments, mice were anesthetized with an intraperitoneal injection of Ketamine-Xylazine (120mg/kg Ketamine, 24 mg/kg Xylazine) and transcardially perfused with PBS followed by 10% neutral-buffered formalin. The brain was delicately extricated and stored in 10% neutral-buffered formalin overnight at 4°C. Tissue was mounted onto a stage and sectioned at 100μm using a VT-1000 vibratome (Leica). Sections were mounted and coverslipped using DAPI Fluoromount-G (Southern Biotech #0100–20). Confocal images were acquired using a LSM800 confocal laser scanning microscope (Zeiss) for injection site location verification, cannula placement, and viral expression in POm axons within pDLS and stereotypical POm-cortical projections in S1 L1 and L5a of all experimental mice.4 (link),25 (link),26 (link),47 (link),49 (link) All data were acquired using the Zen software suite (Zeiss).
Following all behavioral experiments, mice were anesthetized with an intraperitoneal injection of Ketamine-Xylazine (120mg/kg Ketamine, 24 mg/kg Xylazine) and transcardially perfused with PBS followed by 10% neutral-buffered formalin. The brain was delicately extricated and stored in 10% neutral-buffered formalin overnight at 4°C. Tissue was mounted onto a stage and sectioned at 100μm using a VT-1000 vibratome (Leica). Sections were mounted and coverslipped using DAPI Fluoromount-G (Southern Biotech #0100–20). Confocal images were acquired using a LSM800 confocal laser scanning microscope (Zeiss) for injection site location verification, cannula placement, and viral expression in POm axons within pDLS and stereotypical POm-cortical projections in S1 L1 and L5a of all experimental mice.4 (link),25 (link),26 (link),47 (link),49 (link) All data were acquired using the Zen software suite (Zeiss).
10
Mitochondria Localization by AOX Antibody
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N. gruberi was seeded onto sterile poly-L-lysine treated glass coverslips in a 6-well plate and incubated overnight at 25 °C. The following day the cells were treated with 250 nM MitoTracker Red for 30 minutes. The media was then aspirated and cells were washed with 1x PBS, followed by fixation using 2% formaldehyde for 20 minutes. After fixation, 2% formaldehyde was removed and cells were permeabilised with 0.1% triton-X 100 for 10 minutes. After permeabilization cells were washed three times with 1x PBS and blocked using 3% bovine serum albumin in PBS for 1 hour. Primary antibody staining was carried out at room temperature for 1 hour with custom made AOX antibodies (Eurogentec; Peptide: 1911009, Rabbit 237), diluted to 1:1000. Secondary antibody staining was carried out for 1 hour in the dark using anti-Rabbit-IgG-Alexa 488. Slides were washed and mounted using Prolong Gold Antifade with DAPI. Laser Confocal Microscopy was carried out using the LSM 880 Laser Confocal with Airyscan by Zeiss. Laser sets used were 405, 488 and 594, with airyscan detector plate imaging for high resolution. Images were captured and analyzed using Zen software suite by Zeiss.
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