The cultured cochleae were processed for immunofluorescence with standard methods. Prior to the immunoreactions, all the cochleae were first treated for 30 min at room temperature with blocking solution (5% normal donkey and bovine serum in 0.05% Triton X-100 in PBS, pH 7.4). The cochleae were then incubated with the primary and secondary antibodies for each 2 h at room temperature. The used primary and secondary antibodies included: rabbit anti-Myosin7a (1:500, Proteus BioSciences, 25–6790), goat anti-Sox2 (1:300, Santa Cruz Biotechnology, sc-17320), rat anti-BrdU (1:2000, ABD Serotec, OBT0030CX), Alexa 647-conjugated donkey anti-rabbit IgG for Myosin7a labeling (cat. 711-606-152), DyLight 488-conjugated bovine anti-goat IgG for Sox2 labeling (cat. 805-545-180), and Alexa 594-conjugated donkey anti-rat IgG for BrdU labeling (cat. 712-585-150). The specificity of the antibodies has been declared in the protocols and proved in our preliminary experiments. All secondary antibodies were obtained from Jackson ImmunoResearch and diluted at 1:400. After the cochleae were sealed with Antifade Solution (Applygen), they were examined under an inverted Zeiss Axio Observer Z1 fluorescence microscope (20×) and an inverted Zeiss LSM700 laser-scanning confocal microscope (40×). Images were obtained using AxioVision 4.8 and Zen Light Edition programs.
Antifade solution
Manufactured by Applygen
Sourced in China
Antifade solution is a laboratory reagent used to preserve fluorescent signals in biological samples during microscopy or other imaging applications. It helps prevent the fading or quenching of fluorescent dyes or proteins, allowing for more accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti sox2, by Santa Cruz Biotechnology (1 mentions)
Axio observer z1 fluorescence microscope, by Zeiss (1 mentions)
Obt0030cx, by Bio-Rad (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Facsaria flow cytometry, by BD (1 mentions)
Tcs sp2, by Leica camera (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
5 protocols using antifade solution
1
Immunofluorescence Labeling in Cultured Cochleae
2
Assessing Mitochondrial Membrane Potential in HUVEC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential in HUVEC was measured by Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, Shanghai, China). When mitochondrial membrane potential is high in living cells, JC-1 accumulates in matrix in the form J-aggregates presenting red fluorescence; while mitochondrial membrane potential is low in apoptotic cells, JC-1 cannot aggregate, and the JC-1 monomer presents green fluorescence. Decreased mitochondrial membrane potential suggests apoptosis. Briefly, JC-1 working solution and cell medium were mixed at the ratio of 1 : 1. Then, the slides seeded with HUVEC were incubated with the mixture for 20 min at 37°C in the dark. The cells were fixed in prewarmed 4% paraformaldehyde at room temperature for 20 min after washing with ice-cold JC-1 buffer solution twice. After washing three times with PBS, the antifade solution (Applygen, Beijing, China) was used to mount the slides. Analysis was conducted immediately using a laser confocal scanning microscope.
3
Investigating Coumarin-6 Liposomal Uptake in Keratinocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HKFs were inoculated in a plate with 6 wells, adjusted to 1.5×104 cells/well and cultured for 24 hours. Fresh medium containing coumarin-6 (C6) or coumarin-6-loaded liposomes (C6-L) (concentration of C6=1 µg/mL) was added to substitute the previous medium. After incubation for 15 or 30 minutes00, excess C6 and C6-L were rinsed 3 times with PBS, and then cells in wells were collected and analyzed for fluorescence intensity using a FACSAria flow cytometry (BD, Franklin Lakes, NJ, USA).
Simultaneously, in order to observe the fluorescence intensity in the cells, HKFs were seeded in wells with coverslips, and were incubated with C6 or C6-L for 30 minutes after 24 hours. The excess C6 or C6-L were rinsed with cold PBS, and then 4% paraformaldehyde solution was used to fix the coverslips for 10 minutes. Afterward, antifade solution (Applygen, Beijing, China) was dropped onto slides and coverslips were placed on them. Finally, the slides were observed and photographed with 488 excitation wavelength under a confocal laser scanning microscope (TCS-SP2, Leica, Germany).
In order to determine the endocytosis mechanism of PTXL, methyl-β-cyclodextrin (M-β-CD) (caveolin inhibitor) and chlorpromazine (CPZ) (clathrin inhibitor) were used as endocytosis inhibitors to pretreat cells for one hour and removed before adding C6-L. The rest of the processing was the same as above.
Simultaneously, in order to observe the fluorescence intensity in the cells, HKFs were seeded in wells with coverslips, and were incubated with C6 or C6-L for 30 minutes after 24 hours. The excess C6 or C6-L were rinsed with cold PBS, and then 4% paraformaldehyde solution was used to fix the coverslips for 10 minutes. Afterward, antifade solution (Applygen, Beijing, China) was dropped onto slides and coverslips were placed on them. Finally, the slides were observed and photographed with 488 excitation wavelength under a confocal laser scanning microscope (TCS-SP2, Leica, Germany).
In order to determine the endocytosis mechanism of PTXL, methyl-β-cyclodextrin (M-β-CD) (caveolin inhibitor) and chlorpromazine (CPZ) (clathrin inhibitor) were used as endocytosis inhibitors to pretreat cells for one hour and removed before adding C6-L. The rest of the processing was the same as above.
4
MPTP Opening in HUVEC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell MPTP Assay Kit (Genmed, Shanghai, China) was used to test the MPTP opening in HUVEC. Experiment rationale was that calcein-AM could gather in mitochondrion presenting green fluorescent staining, whereas, being released into the cytoplasm via the opening of MPTP, fluorescent quenching could occur. Culture medium was discarded and slides were rinsed gently with cleaning solution. Slides were subsequently incubated with staining working solution for 30 min at 37°C, protecting from light. 4% paraformaldehyde was used to fix the cells for 15 min after rinsing for three times with cleaning solution. Finally, the slides were mounted with antifade solution (Applygen, Beijing, China) after washing with PBS three times and were immediately examined using a laser confocal microscope.
5
Mitochondrial Superoxide Detection in HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher, MA, USA) was used to detect the generation of mitochondrial superoxide anion in HUVEC and MitoTracker Green FM (Thermo Fisher, MA, USA) was used to label mitochondria. The HUVEC on slides were covered with MitoSOX (5 μmol/L) and MitoTracker probes (100 nmol/L) after washing with PBS. The slides were incubated for 20 min at 37°C, avoiding light. Then, the HUVECs were fixed in prewarmed 4% paraformaldehyde at room temperature for 15 min after washing with warm PBS for three times. The slides were mounted by antifade solution (Applygen, Beijing, China) after washing with PBS. Cells on the slides were detected by a laser scanning confocal microscope. Red fluorescence indicated mitochondrial superoxide anion and green fluorescence indicated labeled mitochondria.
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!