HepG2 cells were loaded with MitoTracker Red (500 nM, Cell Signaling Technology, USA) to label the mitochondria as described [29 (link)]. Briefly, after treated as indicated, the HepG2 cells were loaded with MitoTracker Red and imaged by laser confocal scanning microscopy (Zeiss, Germany) under the excitation (Ex) and emission (Em) wavelength of 644 nm and 665 nm, respectively. To observe the mitochondrial fusion, HepG2 cells were labeled with MitoTracker Red and then fixed with 4% paraformaldehyde (Beyotime, China), permeabilized with 0.25% Triton X-100 (Beyotime, China) and blocked with 5% goat serum (Beyotime, China). Then, cells were incubated with primary antibody against OPA1 at 4 °C overnight and incubated with Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (1:200, Beyotime, China). Finally, nuclei were counterstained with DAPI. Samples were analyzed using laser confocal scanning microscopy (Zeiss, Germany).
Confocal laser scanning microscopy
Manufactured by Zeiss
Sourced in Germany, United States, China, United Kingdom, Japan
Confocal laser scanning microscopy is a high-resolution imaging technique that uses a focused laser beam to scan a sample point-by-point. The reflected or fluorescent light is detected and reconstructed into a detailed digital image, allowing for the observation of fine structures and thin optical sections within a specimen.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
In situ cell death detection kit, by Roche (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
G9023, by Merck Group (3 mentions)
Fluorescent in situ hybridization kit, by RiboBio (3 mentions)
Triton x 100, by Beyotime (2 mentions)
Fitc conjugated goat anti mouse igg, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Immunol staining blocking buffer, by Beyotime (2 mentions)
Fluoromount, by Southern Biotech (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Vectashield mounting media with dapi, by Vector Laboratories (2 mentions)
Alexa fluor 488 conjugated anti mouse and 594 conjugated anti rabbit secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Fish kit, by RiboBio (2 mentions)
Fitc labeled circ tgfbr2 probes, by GenePharma (2 mentions)
Cy3 labeled mir 29a probes, by GenePharma (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
211 protocols using confocal laser scanning microscopy
1
Visualizing Mitochondrial Dynamics in HepG2 Cells
2
Immunofluorescence Labeling of Recombinant Yeast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence labeling of recombinant yeast cells was performed to confirm the expression and surface display of Co1-scEDIII-AGA through a slightly modified method, as described previously [36 (link), 39 (link)]. Cultivated recombinant yeast cells (48 h) were washed three times with phosphate-buffered saline (PBS). The pellet was resuspended in PBS containing 1% bovine serum albumin (BSA) and incubated for 30 min at room temperature. Cells were then washed three times with PBS, resuspended in PBS containing 3% BSA and polyclonal anti-dengue mouse IgG (1:100), and incubated at room temperature for 2 h. Next, cells were washed as described above, the pellet was resuspended in PBS containing 3% BSA and secondary antibody, FITC-conjugated goat anti-mouse IgG (1:50; Sigma-Aldrich), and incubated at room temperature for 1 h. After washing, the cells were observed using confocal laser scanning microscopy (Carl Zeiss, Zena, Germany).
3
Immunofluorescence Staining of Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells transfected with indicated plasmids were grown on cover slips. And 48 h later, cells were washed with PBS, fixed with 4% PFA (Sigma), and permeabilized with 0.5% Triton X-100 in PBS and then fixed with antibodies. Nonspecific antibody binding was minimized by treatment with 5% donkey serum in PBS for 30 min at RT. Primary antibodies were diluted in 0.1% Triton X-100 and incubated with the cells for 1 h at 37 °C. Cells were washed three times in PBS and then incubated for 1 h at RT with secondary antibodies conjugated to Cy3 or Cy5 fluorophores (Invitrogen). The cells were then washed three times in PBS and mounted using the anti-fade mounting solution (Dako) and then examined by confocal laser scanning microscopy (Zeiss).
4
Cellular Uptake and Localization of Enrofloxacin Nanogels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RAW 264.7 cells (1 × 105 cells/cm2) were seeded on a 24-well cell culture plate. After 24 h, the cells were incubated with the same concentration (4 μg/mL) of native enrofloxacin and composite nanogels for 0.5, 1, 2, and 4 h. After washing three times with cold phosphate-buffered saline (PBS), the adherent cells were lysed using 150 μL of RIPA cell lysate. The protein content and the concentration of enrofloxacin in the cells were detected using the BCA method and HPLC, respectively.
Simultaneously, the RAW 264.7 cells (1 × 105/dish) were transferred to a confocal dish. After incubation for 24 h, the FITC-labeled enrofloxacin nanogels with the same fluorescence intensity were transferred to the RAW 264.7 cells and incubated for 2 h. After washing three times with PBS, the RAW 264.7 cells were then stained with DiI and Hoechst 33342 for membrane and nuclear staining. The cells were visualized by confocal laser scanning microscopy (Zeiss, Germany) to observe the location of the nanogels in the RAW 264.7 cells.
Simultaneously, the RAW 264.7 cells (1 × 105/dish) were transferred to a confocal dish. After incubation for 24 h, the FITC-labeled enrofloxacin nanogels with the same fluorescence intensity were transferred to the RAW 264.7 cells and incubated for 2 h. After washing three times with PBS, the RAW 264.7 cells were then stained with DiI and Hoechst 33342 for membrane and nuclear staining. The cells were visualized by confocal laser scanning microscopy (Zeiss, Germany) to observe the location of the nanogels in the RAW 264.7 cells.
5
Assessing Viral Infectivity via Direct Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Direct immunofluorescence assay was used to assess the cells’ infectivity to virus infection. The fluorescent signal was tagged on the primary antibody in direct immunofluorescence. Cells were cultured in 6-well plates. MDBK-N4 and MDBK cells were infected with CDV with an MOI (Multiplicity of Infection) of 0.1, and MDBK-TfR and MDBK were infected with CPV at an MOI of 0.1. When cells began to appear CPE, they were fixed with 4% formaldehyde for 10 min at 4 °C followed by pre-chilled PBS wash; cells were treated with 0.3 % Triton-X for 10 min and washed again using pre-chilled PBS. MDBK-N4, MDBK and MDCK cells were incubated with the CDV antibody, and MDBK-TfR and MDBK and CFK cells were incubated with the CPV antibody for 12 h. Fluorescence was detected using confocal laser scanning microscopy (Zeiss).
6
Tubulosine Modulates IL-6-Induced Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at a density of 5 × 104 cells per well in a 12-well plate and then incubated for 48 hours with the vehicle alone or tubulosine in the presence or absence of IL-6. To perform the TUNEL assay, cells were stained using an In Situ Apoptosis Detection kit (Clontech, Mountain View, USA) according to the manufacturer's protocol, and fluorescein isothiocyanate signals were visualized using confocal laser scanning microscopy (Carl Zeiss).
7
Quantifying DNA Double-Strand Breaks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transferred into confocal dishes. Then 4% paraformaldehyde was used for fixation, 0.5% Triton X-100 was used for permeabilization, and 3% BSA was used for blocking. Anti-γH2AX (1:50, D17A3, Cell Signaling Technology [CST], Danvers, MA, USA) antibodies were added at 4 °C overnight. Then, the sample was incubated with DyLight 488 AffiniPure Goat Anti-rabbit IgG (1:200, Abbkine, Redlands, CA, USA) for 1 h in the dark at room temperature, and 4’,6-diamidino-2-phenylindole (DAPI) was used for costaining. Cells were examined using confocal laser scanning microscopy (Zeiss, Germany) and the number of foci of γH2AX (marker of DSB) per nucleus was determined. An average of 100 nuclei per sample were examined.
8
Membrane Permeability Assay of Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effect of peptide on membrane permeability was evaluated using LIVE/DEAD BacLight™ bacterial viability kits (Thermo Fisher, USA) according to the manufacturer’s instructions. Briefly, S. aureus and P. aeruginosa in the logarithmic phase were harvested, and the cells were washed three times and resuspended in NaPB to a final density of approximately 1 × 107 CFU ml-1. The prepared bacteria were then added to the wells to incubate with Spgillcin177–189 (1 × MIC) at 37°C for 30 min. After incubation, the bacteria were harvested and washed twice. SYTO 9 and PI were added to each sample according to the instructions, and the samples were incubated at room temperature in the dark for 15 min. Finally, fluorescent images were obtained using confocal laser scanning microscopy (Zeiss, Germany). Meanwhile, the samples were analyzed immediately by flow cytometry (CytoFLEX, Beckman, USA).
9
Spatial Expression of LINC00853 and FOXP3 in GC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FISH test was applied to examine the localization of LINC00853 and FOXP3 in GC cell lines of MGC803 and MKN-78. Briefly, Specific probes to LINC00853 and FOXP3 were prepared and labeled with cy3 (GenePharma, China) and FITC (GenePharma, China), respectively. After mixing the probes with the pre-made hybridization buffer, samples were incubated overnight in this buffer. Finally, DAPI was applied to counterstain cell nuclei. The images were captured via a confocal laser-scanning microscopy (Zeiss, Jena, Germany).
10
Osteogenic Differentiation of Cell-laden Microcarriers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell‐laden microcarriers were harvested on Day 21 of osteogenic differentiation in a bioreactor, fixed overnight in 4.0% paraformaldehyde, cut into 8 μm thick sections and permeabilized with 0.3% Triton X‐100 for 10 min and blocked with 2% bovine serum album (BSA) for 60 min at room temperature. Then, the sample sections were stained with 10 μg/mL mouse anti‐osteocalcin antibody (R&D systems, USA) overnight at 4°C. The aggregates sections were washed three times with PBS buffer and then incubated with 1:200 Fluorescein (Cy3) donkey anti‐mouse IgG (H + L) (Jackson ImmunoResearch, USA) secondary antibodies in 2.0% BSA for 60 min at room temperature in the dark. Next, the nuclei were stained with Hoechst 33342 and were imaged under confocal laser scanning microscopy (Zeiss, Germany).
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!