An AAV-C1V123 (link) and an AAV-GCaMP6s54 (link) were infused into the DMS. Whole-cell recordings were made in C1V1-expressing neurons. The GCaMP6s measures the calcium signal that is induced by current injection (iPSD), or optogenetic depolarization (oPSD) (Supplementary Figure 2 ). In supplementary Figures 3f–g , fluorescent Ca2+ signals were elicited by eHFS or eHFS+oPSD without whole-cell recording. The distal dendrite (~120 μm from the soma) was chosen for analysis. Ca2+ signals were acquired and analyzed with the Zen program (Zeiss) and Origin software (Origin Lab Corporation, MA), and calculated as previously described55 (link). The fluorescence signals were quantified by measuring the mean pixel intensities of the circular regions of interest (ROI). Fluorescence intensity is expressed as ΔF/F values vs. time, where F is the baseline fluorescence and ΔF is the baseline-subtracted fluorescence.
Zen program
Manufactured by Zeiss
Sourced in Germany
The ZEN program is a software suite developed by Zeiss for the control and analysis of microscopy data. It provides a comprehensive platform for image acquisition, processing, and analysis across a range of Zeiss microscopy systems.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Origin software, by OriginLab (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Matrigel precoated transwell plates, by Corning (1 mentions)
Ultra low attachment 96 well plate, by Corning (1 mentions)
Anti fancd2, by Novus Biologicals (1 mentions)
Axiovert lsm780 microscope, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
17 protocols using zen program
1
Optogenetic Calcium Imaging in Neurons
2
Visualizing Curcumin Uptake in Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the treatment of neurons with curcumin and CurcuEmulsome, the neurons were investigated under a confocal laser scanning microscope (CLSM) periodically at 24, 48 and 72 hours. Uptake of curcumin and CurcuEmulsomes by the neurons was tracked and cell morphology was studied for the presence of any alterations. Accordingly, at DIV2 (day in vitro 2) following the cell seeding, cells were treated with 5 µM free curcumin (curcumin dissolved in DMSO) and 5 µM CurcuEmulsomes. Curcumin and CurcuEmulsome uptake was visualized with the help of the autofluorescent property of curcumin for 72 hours.
Cellular uptake was further studied by three-dimensional (3D) Z-stack imaging via the addition of fluorescent agents at DIV2 after cell seeding. Red Vybrant Tongue (V22885; Thermo Fisher Scientific Orange Red) was used to stain entire cells and Hoechst 33342 (H3570; Thermo Fisher Scientific) (3:1000) was used to stain nuclei. Following 10 minutes of staining, CurcuEmulsomes were added to the culture and images were taken in series for 15 minutes from different Z-stacks with 1-µm intervals. When the imaging was complete, images were merged to realize the 3D structure using the ZEN program (Zeiss).
Cellular uptake was further studied by three-dimensional (3D) Z-stack imaging via the addition of fluorescent agents at DIV2 after cell seeding. Red Vybrant Tongue (V22885; Thermo Fisher Scientific Orange Red) was used to stain entire cells and Hoechst 33342 (H3570; Thermo Fisher Scientific) (3:1000) was used to stain nuclei. Following 10 minutes of staining, CurcuEmulsomes were added to the culture and images were taken in series for 15 minutes from different Z-stacks with 1-µm intervals. When the imaging was complete, images were merged to realize the 3D structure using the ZEN program (Zeiss).
3
Optogenetic Calcium Imaging in Neurons
4
Immunofluorescence Staining and Mitochondrial Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence staining, cells were fixed with 4% PFA in PBS for 30 min at room temperature, washed with PBS, and permeabilized with 0.2% Triton X‐100 in PBS for 5 min on ice. Next, the cells were incubated at room temperature with the primary antibody for 60 min and then with Alexa488‐conjugated secondary antibody for 60 min. For staining with MitoTracker Deep Red FM (M22426, Invitrogen), the culture medium was removed and replaced with pre‐warmed (37°C) medium containing MitoTracker (dilution factor, 1:2000) and incubated at 37°C for 30 min. The cells were wash and fixed with 4% PFA in PBS for 30 min at room temperature. DNA was counterstained with 1 mg/ml 4, 6‐diamino‐2‐phenylindole for 5 min, followed by washing with PBS for 5 min. Cells were imaged using a Zeiss LSM780 FCCS laser confocal microscope. Images were analyzed using the ZEN program (Carl Zeiss) and ImageJ software (National Institutes of Mental Health).
5
Measuring Cell Invasiveness Using Transwell Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell invasiveness was measured using Matrigel-precoated Transwell plates (8-μm pore size; 3422, Corning Costar, NY, USA). Control and shSOX9 cells were seeded at 1 × 105 cells onto these plates, whereas MDAH 2774 cells were transfected with human RIPK1 siRNA of control siRNA for 48 h, trypsinized, counted, and added to the upper chambers of the Transwell plates. Medium containing 10% FBS was added to the lower chambers and acted as the chemoattractant. After incubation for 24 h, the filter was gently removed from the chamber, and noninvasive cells on the upper surface were removed by wiping with a cotton swab. The cells that invaded the Matrigel and attached to the lower surface of the filter were fixed in 100% methanol and stained with 0.1% crystal violet. These were captured at ×200 magnification under a light microscope (BX51, Olympus) and invaded cells were counted using the ZEN program (Zeiss). Data were expressed as the mean ± SD from three independent experiments.
6
Assessing Epidermal Hyperplasia and Immune Cell Infiltration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vertical distances between the basal lamina and outer stratum granulosum were measured to evaluate epidermal hyperplasia. Three random measurements were made per slide using the Zen program (ZEISS, Jena, Germany). Cells were enumerated using a cell-counting grid (2.1 × 1.6 mm, 100× magnification) to evaluate immune cell infiltration in four randomly selected, non-overlapping regions per slide. Immune cells were defined as macrophages, polymorphonuclear leukocytes, lymphocytes, eosinophils, plasma cells, and giant cells [37 (link)]. The number of infiltrated immune cells was represented as the number per square millimeter.
7
TSPO Deletion Induces Intracellular Calcium Changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We assayed TSPO deletion-induced changes of [Ca2+]c via imaging the florescence intensity of Fluo-3AM (1 µM for 60 min) (#F-1242, Thermo Fisher Scientific GmbH, Dreieich, Germany) in the ARPE19 previously described [30 (link),31 (link)]. For the stimulation of the Fluo-3AM in the cells, argon laser at 488 nm in the LSM 800 confocal microscope setup (Zeiss, Ankara, Turkey) with 40 × 1.3 oil objective was used. Before the H2O2 stimulation, Fluo-3AM from the cells was removed by washing with extracellular buffer. The fluorescence intensity changes in the cells were analyzed in a computer by using the ZEN program (Zeiss). Arbitrary unit (a.u.) was used for the expression of the Fluo-3 results.
8
Spheroid Formation of 2774 Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells of type 2774-vec and 2774-sh-SOX9 were cultured in defined serum-free medium composed of DMEM: F12 medium (1:1 mixture, 11320, GIBCO) with the addition of 10 ng/mL of epidermal growth factor (EGF) (236-EG, R&D Systems, Minneapolis, MN, USA), 10 ng/mL of basic fibroblast growth factor (bFGF) (233-FB, R&D Systems), and B27 supplement (17504, GIBCO). The cultured cells were plated in an Ultra-Low Attachment 96-well plate (3471, Corning, Somerville, MA, USA). After 14 days of incubation, spheroids were captured using a microscope (TE2000U, Olympus, Tokyo, Japan), and spheroid number and area were determined by the ZEN program (Zeiss).
9
FANCD2 Foci Quantification by Confocal Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal microscopy was performed as described previously 11 . Briefly, U2OS cells were grown on coverslips (18 mm in diameter; Marienfeld Superior) in 12‐well plates, fixed in 3.7% formaldehyde in PBS for 20 min, permeabilized with 0.2% Triton X‐100 in PBS for 20 min, and blocked with 1% bovine serum albumin in PBST. U2OS cells on coverslips were sequentially incubated with anti‐FANCD2 (Novus Biologicals) overnight and Alexa 488‐conjugated donkey anti‐rabbit secondary antibody for 2 h. After extensive washing with PBST [Phosphate Buffered Saline supplemented with 0.2% Tween‐20], cells were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI), mounted on glass slides, and observed under a Zeiss Axiovert LSM780 microscope (Carl Zeiss, Oberkochen, Germany). To count the number of foci, images were adjusted consistently by changing the gamma and white (brightness) parameters in the ZEN program (Carl Zeiss). After this adjustment, foci with pixel values of over 30 were counted.
10
Confocal Imaging of Angiogenesis and Lymphangiogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal microscopy was used to observe the sprouting, spread, and regression of new lymphatic and blood vessels at days 1, 7, 15, 21, and 28. Samples were imaged at room temperature. Z-stack confocal images obtained using the Zen program (Zeiss, Germany) provided detailed views of vessels growing out in different layers and were used for measurements. Adobe Photoshop (Adobe, San Jose, CA) was used to quantitate branch numbers and measure branch lengths. In addition, outgrowths of fibroblasts were easily excluded because they lacked fluorescence.
To determine the effects of individual growth factors on angiogenesis and lymphangiogenesis, the aortic ring and thoracic duct assays were conducted by culturing tissues in six different endothelial cell growth media formulations (EGM-2 based). The control endothelial cell growth medium included the following: Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Insulin-Like Growth Factor (IGF), VEGF-A, and VEGF-C. The remaining growth media were each deprived of one of the growth factors (growth medium including all growth factors except FGF, etc.). The number of branches and branch length were evaluated on days 1, 7, 15, 21, and 28 and compared with the outgrowth of both vessel types in control endothelial cell growth medium to assess the effect of the absence of a specific growth factor on vessel growth.
To determine the effects of individual growth factors on angiogenesis and lymphangiogenesis, the aortic ring and thoracic duct assays were conducted by culturing tissues in six different endothelial cell growth media formulations (EGM-2 based). The control endothelial cell growth medium included the following: Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Insulin-Like Growth Factor (IGF), VEGF-A, and VEGF-C. The remaining growth media were each deprived of one of the growth factors (growth medium including all growth factors except FGF, etc.). The number of branches and branch length were evaluated on days 1, 7, 15, 21, and 28 and compared with the outgrowth of both vessel types in control endothelial cell growth medium to assess the effect of the absence of a specific growth factor on vessel growth.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!