Autophagy is characterized by the development of autophagic vacuoles. Monodansylcadaverine (MDC) has been proposed as a tracer for autophagic vacuoles as described previously11 (link). HUVECs were cultured on poly-L-lysine-coated cover slips overnight, then treated with different stimuli as described above, and rinsed with PBS. They were then stained with 50 μM MDC at 37 °C for 1 h. After incubation, the cells were fixed for 15 min with ice-cold 4% paraformaldehyde at 4 °C, washed twice with PBS. Images were viewed and captured blindly by two observers using Leica TCS SP5 confocal laser microscopy. The formation of acidic vesicular organelles is related to cell autophagy45 (link). Acridine orange–stained cells fluoresce diffuse green fluorescence, whereas the acidic compartments including autophagic vacuoles show bright red. Cells treated by different stimuli were incubated for 15 min with acridine orange (10 μM in medium from a 10 mM stock in water), washed with PBS, and then examined under Leica TCS SP5 confocal laser microscopy.
Tcs sp5 laser confocal microscopy
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5 is a high-performance laser confocal microscopy system. It provides advanced imaging capabilities for a wide range of applications in life sciences research. The system utilizes a broadband white light laser and multiple detection channels to capture detailed, high-resolution images of samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Tritc labeled goat anti rabbit igg, by Merck Group (2 mentions)
Slowfade reagent, by Thermo Fisher Scientific (2 mentions)
Zetasizer 3000hs, by Malvern Panalytical (1 mentions)
Ckx41 light microscope, by Olympus (1 mentions)
Cary 50, by Agilent Technologies (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Diamidine 2 penylindole dapi, by Merck Group (1 mentions)
Streptavidin conjugated alexa 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
10 protocols using tcs sp5 laser confocal microscopy
1
Fluorescence Microscopy for Autophagy
2
Immunofluorescence Assay for CXCR4 Expression
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SCC-15 cells in the logarithmic phase were digested with 1 ml 0.25% trypsin for 5 min and then centrifuged at 200 x g for 3 min at 4˚C. Subsequently, cells were inoculated (5x104 cells/well) into 6-well plates with coverslips. Cells were cultured in DMEM-12 medium and incubated overnight with 5% CO2 at 37˚C. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS 3 times and blocked with goat serum blocking solution (1% BSA; Beyotime Institute of Biotechnology) at 37˚C for 30 min to block non-specific antibody binding. Subsequently, rabbit anti-human CXCR4 primary antibody (1:200) was added to the cover glass, which was incubated at 4˚C overnight. After washing 3 times with PBS, FITC-labelled goat anti-rabbit IgG secondary antibody (1:100; A0562; Beyotime Institute of Biotechnology) was added to the cells and incubated at 37˚C for 50 min in the dark. After rinsing 3 times with PBS, cells were stained with DAPI for 5 min at room temperature, washed 3 times with PBS and stored at -20˚C until observation. Cells were observed under a TCS-SP5 laser confocal microscopy (magnification, x200; Leica Microsystems GmbH).
3
Comprehensive Analysis of PLGA Nanoparticles
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Samples were gently stirred with a pipette until they were fully dispersed and assessed for surface morphology, distribution and size using a CKX41 light microscope (magnification, x100); Olympus Corporation) and a JEOL JSM-7800F scanning electron microscope (magnification, x2,000; Hitachi, Ltd.). A drop of the nanoparticles (50 ug/ml) was placed on a silicon panel (5x5 mm) until it dried naturally in the dark overnight at room temperature. The silicon panel then analyzed via scanning electron microscopy. A TCS-SP5 laser confocal microscopy (magnification, x400; Leica Microsystems GmbH) was used to observe SDF-1 conjugation. A laser particle size meter (Zetasizer 3000HS; Malvern Instruments, Inc.) was used to detect particle size and Zeta potential. Determination of the iron content in the samples (3 (link)) was performed via graphite furnace atomic absorption spectrometry. The wavelength used for detection was 248.3 nm. The absorption spectrum of the nanoparticles was detected using an ultraviolet-visible (UV-Vis) spectrophotometer (CARY 50; Varian Medical Systems) and blank PLGA nanoparticles were used as the control group.
4
Immunofluorescence Analysis of TGEV Infection in ST Cells
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ST cells inoculated with TGEV were cultured for 24 h. The cells were washed twice with PBS and fixed with paraformaldehyde (4%) for 30 min at 4 °C, and then allowed to air dry. After blotting with 5% skimmed milk powder, the fixed cells were incubated with mAb to TGEV N protein (1:100) and rabbit pAb to EF1A (1:50) for 1 h at 37 °C in a humidified chamber. After washing three times with PBST, the fixed cells were incubated with FITC-labeled goat anti-mouse IgG (1:100, KPL) and TRITC-labeled goat anti-rabbit IgG (1:200, Sigma). The additional nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was performed as described previously (Jungmann et al., 2001 (link)). The triple-stained cells were washed three times with PBST and subsequently examined under a Leica TCS SP5 laser confocal microscopy.
5
Immunohistochemical Staining of Ileal Tissue
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Frozen sections of ileal tissue were fixed with 4% paraformaldehyde for 10 min at room temperature, then washed thrice with PBS, permeabilized in 0.1% Triton X-100 in PBS for 5 min at room temperature. After washing with PBS, the nonspecific binding sites were blocked with 5% normal goat serum in PBS for 30 min. Then, the sections were incubated with monoclonal rabbit antibody against ZO-1 (1:200, Invitrogen), ASC (1:100, Cell Signaling), LC3B (1:100, Sigma) at 4°C overnight. After washing thrice in PBS, sections were incubated with secondary Alexa Fluor 594 conjugated goat anti-rabbit IgG antibody (1:200, Invitrogen) and DAPI (1:1000, Sigma) for 1 hour at room temperature. After washing thrice in PBS, sections were mounted using Slowfade reagents (Invitrogen). Images were obtained using a TCS SP5 laser confocal microscopy (Leica, Germany).
6
Immunofluorescence Assay for ASS1 Protein
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293T cells were seeded in 24-well plates and cultured in an incubator at 37°C and 5% CO2. When the cell density reached 30%–40%, wild-type or mutant ASS1 plasmids were transfected into 293T cells using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific, Shanghai, China). In line with the standard experimental procedure, the cells were harvested after 48 h, fixed with 4% paraformaldehyde at room temperature for 15–20 min, permeabilized with 0.5% TritonX-100 (PBS formulation) for 20 min, and then blocked with 5% bovine serum albumin (BSA) for 30 min. They were then incubated with primary antibody (1:300) in 5% BSA overnight at 4°C. The next day, the cells were incubated with a secondary antibody (Dylight 488 Mouse Anti-Goat antibody) for 1 h and then with DIPA for 10 min, and then shielded using Fluoromount™ Aqueous Mounting Medium (Sigma Aldrich, St. Louis, MO, United States). Finally, TCS SP5 laser confocal microscopy (Leica, Wetzlar, Germany) was used to acquire the immunofluorescence images.
7
Immunofluorescence Imaging of Intestinal Tight Junctions
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We applied the methods which usually utilized and described in previous articles [19 (link)]. Frozen sections of ileum were prepared for detecting ZO-1 morphology. After preparatory work, the sections were incubated with monoclonal rabbit antibody against zonula occludens (ZO)-1 (Invitrogen, Carlsbad, CA) diluted at 1:200 in 5% normal goat serum in phosphate buffer saline (PBS) at 4 °C overnight. Then the sections were incubated with secondary Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen) at 1:200, Alexa Fluor 594-conjugated phalloidin (Invitrogen) at 1:100, and diamidine-2-penylindole (DAPI) (Sigma) at 1:1000 for 1 h at room temperature. Images were obtained using a TCS SP5 laser confocal microscopy (Leica, Germany).
8
Quantifying Adult Neurogenesis via BrdU Immunofluorescence
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For BrdU immunofluorescence, sections were denatured in 2 M HCl in TBS for 30 min, rinsed and incubated with mouse anti-BrdU (1:100, OBT-0030, Oxford Biotechnology, UK) for 2 d. Then sections were rinsed, incubated with biotinylated anti-rat (1:250; Vector) for 90 min, rinsed, and incubated for 30 min in the dark with streptavidin-conjugated Alexa 568 (1:1,000; Molecular Probes) to visualize BrdU. The analysis was performed using confocal images from coronal sections at similar rostro-caudal levels obtained with a Leica TCS SP5 laser confocal microscopy. An experimenter blinded to the treatment groups counted the BrdU+ cells in the subgranular zones and granule cell layers. The positive cells were counted on sets of every sixth section (six sections per rat) through the rostral-caudal extent of the hippocampal DG.
9
Immunofluorescent Staining of Ileal Tight Junctions
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Frozen sections of ileal tissue were fixed with 1% paraformaldehyde in PBS containing 1 mmol/L CaCl2 for 30 min at room temperature, then washed thrice with PBS for 5 min, permeabilized in 1% Triton X-100 in PBS at room temperature for 5 min. After washing with PBS, the non-specific binding sites were blocked with 5% normal goat serum in PBS for 30 min. Then, the sections were incubated with monoclonal rabbit antibody against ZO-1, occludin, claudin-1 or claudin-2 (Invitrogen) diluted at 1:200 with 1% normal goat serum in PBS at 4°C overnight. After washing thrice in PBS, sections were incubated with secondary Alexa Fluor 594 goat anti-rabbit IgG antibody (Life Technologies) at 1:200, Alexa Fluor 488-conjugated phalloidin (Invitrogen) at 1:100, and DAPI (Sigma) at 1:1,000 for 1 h at room temperature. After washing thrice in PBS, sections were mounted using Slowfade reagents (Invitrogen). Images were obtained using a TCS SP5 laser confocal microscopy (Leica, Germany).
10
Visualizing TGEV Infection in ST Cells
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ST cells inoculated with TGEV were cultured for 0, 1, 2, 4, 8, and 16 h. Cells were washed twice with PBS and fixed with paraformaldehyde (4%) for 30 min at 4 °C, and then allowed to air dry. After blotting with 5% skimmed milk powder, the fixed cells were incubated with mAb to TGEV N protein (1:200) and rabbit pAb to vimentin (1:100, Abcam) for 1 h at 37 °C in a humidified chamber. After washing three times with PBST, the fixed cells were incubated with FITC-labeled goat anti-mouse IgG (1:100, KPL) and TRITC-labeled goat anti-rabbit IgG (1:200, Sigma). Additional nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was performed as described previously (Jungmann et al., 2001 (link)). The triple-stained cells were washed three times with PBST and subsequently examined under a Leica TCS SP5 laser confocal microscopy.
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