Co-location of HER3 Ab and aptamer was performed to verify the specificity. MCF-7 cells were seeded on round glass and grown overnight. The cell-filled dishes were washed with PBS and fixed with fresh 2% paraformaldehyde at 25°C for 30 min. Then the cell-filled dishes were washed three times with 1 mL of PBS. The MCF-7 line was incubated with anti-human HER3 Ab and FITC-Apt at the same time at 25°C for 1 h in the dark. Then the dishes were washed three times with PBS. Next, the MCF-7 cells were incubated with the rhodamine-labeled goat anti-rabbit IgG and incubated for 45 min in the dark as secondary antibody against HER3 Ab. After three washes with PBS, DAPI was added to the cells and incubated for 3 min. Finally, the cells were fixed and prepared for visualization by Nikon Ti A1 confocal laser scanning microscopy (Nikon Ti A1; Nikon Corporation, Tokyo, Japan).
A1 ti
Manufactured by Nikon
Sourced in Japan
The A1 Ti is a high-performance benchtop imaging system designed for advanced microscopy applications. It features a titanium-alloy optical path and stage for enhanced stability and vibration control. The A1 Ti supports a range of imaging modalities, including confocal, multiphoton, and super-resolution techniques. Its modular design allows for customization to meet the specific needs of research laboratories and core facilities.
Automatically generated - may contain errors
Lab products found in correlation
Kgaf001, by Keygen Biotech (1 mentions)
Vectastain abc staining kit, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Scn400, by Leica (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Pannoramic scan, by 3DHISTECH (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Alexa fluor 647 conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
Nlrp3, by Cell Signaling Technology (1 mentions)
Caspase 1, by Novus Biologicals (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Sangon (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
14 protocols using a1 ti
1
Visualizing HER3 Aptamer Colocalization
2
Evaluating 3D Bioprinted HUVEC-Laden Scaffolds
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HUVECs were added to four groups of bioinks (0%, 0.5%, 1% and 2% ChiNC), with a cell density of 8 × 106 cells/ml. 3D bioprinted grid scaffolds were cultured in ECM, and cell viability within the 3D bioprinted scaffolds was assessed using a fluorescent live/dead activity assay kit (KGAF001, KeyGEN Bio tech, China) on Days 1 and 7 according to the protocol. The scaffolds were visualized under a fluorescence microscope (NIKON Ti-A1, Japan), and images were processed with Image J software.
3
Immunofluorescence and Immunohistochemistry Protocols
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Slices or fixed cultured cells were permeabilized with 0.5% Triton X-100 and blocked with PBS plus 10% horse serum and 3% bovine serum albumin (BSA). The samples were incubated with primary antibodies (diluted to 1:400 with PBS) overnight at 4 °C. Then, the samples were washed with PBS with Tween 20 (PBST) 4 times and incubated with fluorescently labeled secondary antibodies at 25 °C for 90 min. Finally, the samples were washed once with PBST, the nuclei were stained with 4',6-diamidino-2-phenylindole (#D9542, Sigma-Aldrich), and the samples were washed with PBST a further 3 times. Then, the slices were mounted with ProLong Diamond Antifade Mountant (#P36961, ThermoFisher) and coverslips. Images were captured with a laser confocal microscope (Ti-A1, NIKON, Minato-ku, Tokyo, Japan) and a fluorescent slide scanner (Pannoramic SCAN, 3DHISTECH, Budapest, Hungary). For immunochemistry, samples were pretreated with 0.3% H2O2 in PBS and the secondary antibody was used according to the user's manual of the VECTASTAIN ABC staining kit (#PK-4001, Vector Laboratories, Burlingame, CA, USA). Images were captured using a slide scanner (SCN400, Leica, Buffalo Grove, IL, USA).
4
Cytocompatibility of BASCs in Au@Pt/Alg Hydrogel
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The BADCs were seeded
in different concentrations of Au@Pt/Alg
hydrogel (0, 0.2, 0.5, 1, and 2 mg/mL) for 3 and 7 days culturing.
The samples were treated by live/dead staining for 30 min in the dark
and live and dead cells were detected by confocal laser scanning microscope
(CLSM, Nikon Ti A1) to evaluate the cytocompatibility of BASCs in
Au@Pt/Alg hydrogel. At the same time, the samples’ cultured
medium was replaced with medium containing 10% alamar blue solutions.
Additional 4 h culturing was performed before the absorbance was measured
by a microplate reader (Molecular Devices, at 570).
The intracellular
superoxide anions level was evaluated by a dihydroethidium (DHE) staining
kit. Briefly, BASCs in Au@Pt/Alg hydrogel were cultured for 24 h in
normal or 200 μM H2O2-induced ROS microenvironment.
The samples were incubated with DHE for 15–25 min. Cell nucleuses
were stained by 4′,6-diamidino-2-phenylindole (DAPI) after
washing with PBS and were observed under CLSM.
in different concentrations of Au@Pt/Alg
hydrogel (0, 0.2, 0.5, 1, and 2 mg/mL) for 3 and 7 days culturing.
The samples were treated by live/dead staining for 30 min in the dark
and live and dead cells were detected by confocal laser scanning microscope
(CLSM, Nikon Ti A1) to evaluate the cytocompatibility of BASCs in
Au@Pt/Alg hydrogel. At the same time, the samples’ cultured
medium was replaced with medium containing 10% alamar blue solutions.
Additional 4 h culturing was performed before the absorbance was measured
by a microplate reader (Molecular Devices, at 570).
The intracellular
superoxide anions level was evaluated by a dihydroethidium (DHE) staining
kit. Briefly, BASCs in Au@Pt/Alg hydrogel were cultured for 24 h in
normal or 200 μM H2O2-induced ROS microenvironment.
The samples were incubated with DHE for 15–25 min. Cell nucleuses
were stained by 4′,6-diamidino-2-phenylindole (DAPI) after
washing with PBS and were observed under CLSM.
5
Characterization of BMSC Cell Sheets
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At day 5, fabricated BMSC sheets were fixed by 4% paraformaldehyde and underwent dehydration, embedding, paraffin sectioning, and processing for H&E staining. The structure of the cell sheet was then observed by microscope. After culturing for 5, 7, and 10 days, cell sheets were harvested and fixed by 4% paraformaldehyde for 30 min. FITC-phalloidin dye was used to stain the F-actin of the cell sheet while nuclei were labeled with DAPI. The dye was removed and washed with 1x PBS before observation with confocal microscopy (Nikon A1 Ti, Tokyo, Japan). To determine the thickness of the cell sheet, the z-direction slicing mode was used and an average of nine points was taken.
6
Autophagy Analysis in MCF7/TAMR Cells
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MCF7/TAMR cells stably expressing tandem mCherry-EGFP-LC3 (described previously) were further infected with shCtrl, shH19, shH19+shSAHH, or shH19+shDNMT3B and selected using puromycin for 1 week. Then, the cells were seeded on a coverglass for growth and cultured in medium with 10 μM tamoxifen. After 24 h, the cells were fixed, stained with DAPI (300 nM), and then examined using a confocal microscope (Nikon A1 Ti).
7
Axonal Transport Imaging in Microfluidic Devices
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Microfluidics devices were prepared as described in (41 (link)). Axonal transport recording was performed after 5 DIV, as previously described (16 (link)), using an inverted confocal microscope (Nikon, A1Ti), at a 600-ms interval for 60 s using 60× lens. For cell culture, the recording microscope chamber was heated at 37°C and supplied with 5% CO2.
8
FITC-labeled FK506 Liposome Uptake in HCECs
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1*105 HCECs were seeded into confocal dishes and adhered to for 24 h, the culture medium was then removed, and the cells were washed three times. Hundred μl of FK506 liposome labeled with FITC was co-incubated with cells for 2 h with 100 μl DMEM in the cell incubator. The excess liposomes and DMEM were removed, and the cells were washed three times. The nuclei of the HCECs were stained by DAPI. Confocal microscopy (A1 Ti, Nikon) was used to show the position of FK506 liposomes in cells.
9
Immunofluorescence Staining of Cells
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The cells were treated as previously described (24 (link)). Briefly, cells or tissue slices were incubated overnight at 4°C with primary antibodies (Supplementary Table 3
10
Losartan Enhances Dox-L Tumor Accumulation
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For in vivo Dox-L biodistribution analysis, 4T1 tumor-bearing mice were randomly distributed into two groups (n = 3) for different treatment: Dox-L (control) and Losartan + Dox-L. The 4T1 tumor-bearing mice from the group Losartan + Dox-L were injected intraperitoneally with 200µL 4 mg/mL losartan for 5 days before Dox-L injection. The 4T1 tumor-bearing mice from both groups received injection with Dox-L at a dose of 5mg/kg via the tail vein. Tumors were surgically excised at 12h post-injection to make frozen sections. Staining for blood vessels was done by incubation with rabbit anti-CD31 mouse monoclonal antibody (mAb) (dilution 1:200, Cell Signaling Technology, USA) and Alexa Fluor®647-conjugated anti-rabbit secondary antibody (dilution 1:500, Cell Signaling Technology, USA). DAPI was applied to stain cell nuclei (dilution 1:5000, USA). The images were captured with confocal microscopy (Nikon A1 Ti, Japan).
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