For immunohistochemistry, 3- to 3.5-μm sections of formalin-fixed paraffin-embedded tissue sections were treated with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20) at 95–98 °C for 20 min for antigen unmasking. The anti-HPSE rabbit monoclonal antibody (Abcam; 1:100) was used for immunostaining. Staining patterns were visualised with Histostain-Plus 3rd Gen IHC Detection Kit (ThermoFisher Scientific). The sections were counterstained with Hematoxylin and observed by light microscopy using AxioScopeA1 microscope (Zeiss, Oberkochen, Germany). Quantitative analysis was perform with ZENblue program (Zeiss)
Zenblue program
Manufactured by Zeiss
Sourced in United Kingdom, Germany
ZENblue is a software program developed by Zeiss to control and operate various microscopy and imaging equipment. It serves as a unified platform for managing the functionalities of Zeiss-manufactured lab instruments. The core function of ZENblue is to provide a user-friendly interface for efficiently configuring, capturing, and processing images and data from Zeiss imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Axio scope a1 microscope, by Zeiss (2 mentions)
Axio observer z1 live cell imaging system, by Zeiss (2 mentions)
Cal 520 am, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Rabbit anti trpv1 antibody, by Alomone (1 mentions)
Antibiotic antimycotic mixture, by Merck Group (1 mentions)
Triton x 100 0.1, by Merck Group (1 mentions)
Lsm700 axioobserver confocal microscope, by Zeiss (1 mentions)
M199 medium, by Merck Group (1 mentions)
Cal 520, by Abcam (1 mentions)
7 protocols using zenblue program
1
Immunohistochemical Analysis of HPSE Expression
2
Quantifying Astrocytes and Microglia in Hippocampus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Z-stack images of the hippocampus were acquired on a Zeiss LSM 780 single-photon confocal system using n=4 sections of each genotype. In order to count the number of astrocytes and microglia in the molecular layer and dentate hilus, images were acquired by performing a tile scan at 20x with a Z-stack of 7 slices and 2 µm thickness. The images were stitched in the Zeiss Zen Blue program. We randomly selected 3–4 equal sized areas per tissue on the molecular layer and outlined the area of the dentate hilus in each mouse. The volume of the dentate hilus and molecular layer were calculated by multiplying their respective area by its thickness. The cell count was divided by the resultant section volume to obtain the total cell density in the dentate hilus and molecular layer per mm3.
3
Immunofluorescence Imaging of TRPV1 in Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ECs were grown on coverslips in Petri dishes with M199 medium (Sigma-Aldrich St Louis MO) supplemented with 10% FBS (HyClone Thermo-fisher Waltham Mass USA) and an antibiotic-antimycotic mixture (Sigma-Aldrich St Louis MO) for 48 hrs. at 37°C in an atmosphere with 5% CO2. Subsequently, cells were stimulated with CS for 30 minutes. Then, the cells were fixed with a 4% paraformaldehyde solution for 10 minutes at 4°C. The cells were permeabilized with Triton X-100 0.1% (Sigma-Aldrich St Louis MO) at room temperature for 10 minutes, followed by incubation with a rabbit anti-TRPV1 antibody 1 : 50 (Alomone Labs Jerusalem ISR), in a humidified chamber overnight at 4°C. An unlabeled anti-TRPV1 antibody was detected by incubation with an anti-rabbit anti-IGg antibody conjugated with fluorescein isothiocyanate (FITC) (Thermo Fisher Waltham Mass USA). The cells were examined by confocal fluorescence microscopy with an inverted LSM 700 Axio Observer confocal microscope (Carl Zeiss Jena GmbH) at a 40X magnification. We include a 2.5 X digital zoom image in order to appreciate more cell details. DAPI was used to contra-stain the nucleus. The intensity of fluorescence was determined by the zen blue program (Carl Zeiss Jena GmbH).
4
VSMC Calcium Signaling Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
VSMC Ca2+ signaling was assessed using the fluorescent Ca2+ indicator, Cal‐520 acetoxymethyl ester (Cal‐520/AM; 10 μmol/L; Abcam, Cambridge, UK). Cells were grown in 12‐well plates and following removal of culture media were incubated with Cal‐520 AM in 0.5% FBS at 37 °C for 75 minutes followed by 30 minutes at room temperature. Following incubation, the dye solution was replaced with HEPES physiological saline solution (1.3×10−1 mol/L NaCl, 5×10−3 mol/L KCl, 10−3 mol/L CaCl, 10−3 mol/L MgCl, 2×10−2 mol/L HEPES, and 10−2 mol/L D‐glucose, pH 7.4) for 30 minutes prior to imaging. Fluorescence intensity as a measure of [Ca2+]i, was monitored for 30 seconds in basal condition and 180 minutes under endothelin‐1 (0.1 μmol/L) or U46619 (1 μmol/L) stimulation. In some experiments, VSMCs were pretreated for 30 minutes with olaparib (1 μmol/L) or 8‐Br‐cADPR (1 μmol/L). Fluorescence‐based measurements of Ca2+ signals were performed using an inverted epifluorescence microscope (Axio Observer Z1 Live‐Cell imaging system; Zeiss, Cambridge, UK) with excitation/emission wavelengths 490/535 nm, respectively. Images were acquired and analyzed using the Zen Blue Program (Zeiss, Cambridge, UK).
5
Visualizing Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential was visualized in treated cell stained with JC-1 (Cat# T3168, ThermoFisher) using a confocal microscope (LSM880, Zeiss). Cells were collected, washed in ice-cold PBS and mounted on glass slides using a Cytocentrifuge (CytoSpin4, 600 rpm, 10 min). Cells were then washed with PBS, fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 15 min. Then the cells were stained with JC-1 dye for 1 h at 37 °C. According to the manufacture, JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a green fluorescence emission at (~529 nm) for the monomeric form of the probe, which shifts to red (~590 nm) with a concentration-dependent formation of red fluorescent J-aggregates. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio (ThermoFisher). Quantification of fluorescence intensity was done using Zen Blue program (Zeiss).
6
Immunostaining of Decorin and Brevican
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry, 3- to 4-µm sections of formalin-fixed, paraffin-embedded tissue were deparaffinized, and antigen was retrieved by treatment with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20) at 95–98 °C for 20 min. Non-specific binding blocking, immunostaining and visualization were performed using Mouse- and Rabbit-specific HRP/DAB (ABC) Detection IHC kits (Abcam cat. N ab64264) according to the manufacturer’s instructions. The rabbit polyclonal anti-decorin (Abcam, cat. N ab175404, 1:100) and rabbit polyclonal anti-brevican antibody (Abcam cat. N ab111719, 1:100) primary antibodies were used for immunostaining for 1 h at room temperature. The sections were counterstained with hematoxylin and observed by light microscopy using an AxioScopeA1 microscope (Zeiss, Jena, Germany). Quantitative analysis was performed with the ZENblue program (Zeiss, Jena, Germany).
7
Measurement of Intracellular Calcium Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular free Ca2+ levels were measured in RAEC using the fluorescent Ca2+ indicator, Cal-520 acetoxymethyl ester (Cal-520/AM; Abcam; 10 μmol/l). Fluorescence measurements were performed using an inverted epifluorescence microscope (Axio Observer Z1 Live-Cell imaging system; Zeiss) with excitatory wavelengths of 490 and emission of 535. Images were acquired and analysed using Zen Blue Program (Zeiss). Cells were grown in six-well plates and following the removal of culture media were incubated with 10 μmol/l of Cal-520 AM in 0.5% FBS at 37°C for 75 min followed by 30 min at room temperature. Following incubation, the dye solution was replaced with HEPES physiological saline solution containing the following components (in mmol/l): NaCl 130, KCl 5, CaCl 1, MgCl 1, HEPES 20 and D-glucose 10, pH 7.4) for 30 min before imaging. Fluorescence intensity as a measure of [Ca2+]i, was monitored for 30 s in basal condition and 2.5 min under Ang II 10−7 mol/l stimulation.
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!