Mice were perfused intracardially with ice cold PBS (30 ml) and 4% PFA (15 ml). Intact CNS tissue was dissected and immunohistochemical analysis was performed on 10 μm thick cryosections. For antigen retrieval, the tissue sections were incubated with 10 mM citrate buffer (pH 6.0) for 20 minutes at 70 °C. Afterwards the samples were treated with blocking solution (antibody diluent, Agilent Technologies, #S080983-2) and incubated with anti-CD4 (EPR6855, Abcam, #ab183685, 1:100) at 4 °C for 16-18 h. After incubation with primary antibody, the tissue sections were washed thoroughly with PBST (PBS + 0.05% Tween-20) and incubated with anti–rabbit AF647 (Abcam, #ab150087, 1:500) at 37 °C for 1 h. The sections were washed and stained with FluoroMyelin Red (Invitrogen, # F34652, 1:50) and mounted with ProLong Gold antifade mounting media (Life Technologies, #P36965). The images were acquired at 20x magnification with a Nikon Eclipse Ti2 microscope and NIS Elements AR (v.5.20.00) software or at 20x and 60x magnifications with a Leica SP8 confocal microscope and the Leica Applications Suite X (v.3.5.6.21594) software.
X applications suite
Manufactured by Leica
Sourced in Germany
The X Applications Suite by Leica is a software package designed to operate Leica's laboratory equipment. It provides a unified interface to control and manage various Leica instruments, enabling efficient workflow and data management within the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Dmi8 confocal microscope, by Leica (6 mentions)
Sp8 confocal microscope, by Leica (3 mentions)
Eclipse ti2 microscope, by National Instruments (2 mentions)
Prolong gold antifade mounting media, by Thermo Fisher Scientific (2 mentions)
Fluoromyelin red, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Imagej, by Leica (1 mentions)
Naphthol as d chloroacetate kit, by Merck Group (1 mentions)
Eosin y solution, by Merck Group (1 mentions)
Dmi8 fluorescence microscope, by Leica (1 mentions)
12 protocols using x applications suite
1
Immunohistochemical Analysis of CD4+ Cells
2
Immunohistochemical Analysis of CD4+ Cells
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Mice were perfused intracardially with ice cold PBS (30 ml) and 4% PFA (15 ml). Intact CNS tissue was dissected and immunohistochemical analysis was performed on 10 μm thick cryosections. For antigen retrieval, the tissue sections were incubated with 10 mM citrate buffer (pH 6.0) for 20 minutes at 70 °C. Afterwards the samples were treated with blocking solution (antibody diluent, Agilent Technologies, #S080983-2) and incubated with anti-CD4 (EPR6855, Abcam, #ab183685, 1:100) at 4 °C for 16-18 h. After incubation with primary antibody, the tissue sections were washed thoroughly with PBST (PBS + 0.05% Tween-20) and incubated with anti–rabbit AF647 (Abcam, #ab150087, 1:500) at 37 °C for 1 h. The sections were washed and stained with FluoroMyelin Red (Invitrogen, # F34652, 1:50) and mounted with ProLong Gold antifade mounting media (Life Technologies, #P36965). The images were acquired at 20x magnification with a Nikon Eclipse Ti2 microscope and NIS Elements AR (v.5.20.00) software or at 20x and 60x magnifications with a Leica SP8 confocal microscope and the Leica Applications Suite X (v.3.5.6.21594) software.
3
Time-lapse Imaging of Calcifying Cells
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Light microscopy images were acquired using a DMi8 Inverted Microscope with a DFC700 T colour camera (Leica Microsystems, Milton Keynes, UK). During time‐lapse imaging, cells were placed on a cooled stage at 17°C. For time‐lapse imaging of cell division, cells were maintained in the dark and illuminated only for image capture (exposure, 300 ms; frame rate, 5 min). Approximately 10–20 cells were viewed simultaneously for each time lapse. Where stated, cells were gently decalcified with 0 mM Ca2+ ASW at pH 7.0 for 1 h before resuspension in FSW f/2. To monitor the response to Ge treatment, cells were grown in 40 ml of culture and 1 ml aliquots were removed every 24 h for time‐lapse imaging over a period of 12 h. Cells were maintained on the microscope in constant light to encourage calcification. Approximately 100–120 cells were viewed simultaneously for these time lapses. Images and sequences were processed using Leica Applications Suite X and Image J (Abràmoff et al., 2004 ) software.
4
Histological Assessment of Intestinal and Brain Tissue
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The right hemisphere of the brain and small and large intestine were embedded in paraffin and sections (4 µm, large intestinal sections n = 6/group due to technical issues) were stained with hematoxylin (1 min, Hematoxylin Solution, Gill No. 2, 4 g/L, Sigma Aldrich, Darmstadt, Germany) and eosin (2 sec, Eosin Y solution, alcoholic, 0.5% (w/v) in acidified ethanol, Sigma-Aldrich, Germany). To assess villous atrophy in small intestine, villus height and crypt depth was assessed while crypt depth was also assessed in large intestine using an analysis system (Leica Applications Suite X, Leica, Wetzlar, Germany) integrated in a microscope (Leica DM6 B, Leica, Germany). Neutrophil granulocytes were stained in 4 µm brain sections using Naphthol AS-D Chloroacetate Kit (Sigma-Aldrich, Germany) as described by the manufacturer. For the detection of Nissl bodies in the cytoplasm of neurons, 4 µm brain sections were deparaffinized and hydrated in ethanol. After staining for 10 min in 0.1% cresyl violet solution at 37 °C (diluted in distilled water and 0.3% (w/w) glacial acetic acid) sections were washed and differentiated in 95% ethanol, dehydrated, cleared and mounted.
5
Visualization of Cytokine Signaling in Th17 Cells
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MACS-sorted naive T cells from wild-type mice were cultured in Th17 differentiation conditions. On day 1, T cells (106) were nucleofected with 5 µg of mRuby2-N1-IL-24t or mRuby2-N1-IFN-γt and plated into poly-L-lysine–coated chamber slides (80824; Ibidi). On day 2, cells were treated with Mitotracker green (10 nm, M7514; Thermo Fisher Scientific) for 30 min. Cells were washed and analyzed by confocal microscopy. Images were acquired at 60× magnification using a Leica SP8 confocal microscope and Leica Applications Suite X (v3.5.6.21594) software. Cytokine signal colocalization was quantified using ImageJ (1.52i).
6
Quantifying Spike-Mediated Cell Fusion
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HEK293T-ACE2 cells were co-transfected with GFP and the spike of interest. Cells were imaged 18 h post-transfection using a Leica DMi8 fluorescence microscope. Average area of fused cells was determined using the Leica X Applications Suite software that outlines edges of syncytia and calculates the area within. Three images were randomly taken for each variant. Scale bars represent 150 µM and one representative image was selected for presentation.
7
Syncytia Assay for Viral Fusion
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Syncytia formation was used to determine the ability of the spikes of interest to mediate viral membrane fusion. As described previously, HEK293T-ACE2 cells were co-transfected with the spike of interest and GFP, incubated for 24 h and imaged using fluorescence microscopy (Leica DMi8 confocal microscope) to observe fusion. Two wells were imaged for each sample with two randomly selected images taken. Software within the Leica X Applications Suite was used to quantify the average area of fused cells by outlining the perimeters of syncytia and calculating the area inside. The “No Spike” control refers to a well that was transfected with GFP and empty pcDNA3.1 plasmid backbone to serve as a negative control.
8
SARS-CoV-2 Spike-Mediated Cell Fusion
9
SARS-CoV-2 Spike-Mediated Cell Fusion
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To measure the extent of cell-cell fusion mediated by the different SARS-CoV-2 spikes, HEK293T cells expressing ACE2 were co-transfected with spike plasmid and GFP.6 (link) Cells were imaged with a Leica DMi8 confocal microscope 30-hours post-transfection. Representative images were selected for presentation. Area of fused cells was determined and quantified using the Leica X Applications Suite, scale bars represent 150 μM.
10
Cell-Cell Fusion Assay for S Protein
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Cell-cell fusion assays were carried out as follows. Briefly, HEK293T cells were cotransfected with individual S plus a GFP plasmid; transfected cells were digested next day by trypsin and then cocultured with CaLu-3 cells at a 1:1 ratio. Following 24–48 hrs incubation, the cells were imaged under a Leica DMi8 confocal microscope. The areas of fused cells were quantified using the Leica X Applications Suite.
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