For immunocytochemistry, cells were grown on 35-mm glass-base dishes (Iwaki, Chiba, Japan) coated with rat tail collagen. They were fixed with an ethanol and acetone 1:1 mixture at −20 °C for 10 min. Staining was performed as described previously22 (link) using the primary polyclonal anti-tricellulin antibody and monoclonal anti-occludin antibody. The specimens were examined using an epifluorescence microscope (Olympus, Tokyo, Japan) and a confocal laser scanning microscope (LSM510; Carl Zeiss, Jena, Germany).
35 mm glass base dishes
Manufactured by Iwaki
Sourced in Japan, United States
35 mm glass base dishes are circular glass containers with a diameter of 35 millimeters. They serve as a standard substrate for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Type 4 a, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Epifluorescence microscope, by Olympus (1 mentions)
Ab110767, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ab16056, by Abcam (1 mentions)
Fv10i doc, by Olympus (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Roche (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
Staurosporine, by LKT Laboratories (1 mentions)
Image it live green caspase 3 and 7 detection kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using 35 mm glass base dishes
1
Immunocytochemistry of Tricellulin and Occludin
2
Zebrafish Embryo Imaging and Manipulation
3
Immunofluorescence Imaging of LNCaP Cells
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LNCaP cells were grown in 35 mm glass base dishes (IWA-KI, Chiba, Japan), washed twice with D-PBS, and fixed with 100% MeOH for 20 min at room temperature. Fixed cells were incubated with detector blocking solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) overnight at 4°C. Cells were incubated with 2 μg/mL anti-CTL1 rabbit polyclonal antibody (ab110767, Abcam plc, Cambridge, UK), 2 μg/mL anti-CTL2 mouse monoclonal antibody (clone 2D11, Abnova, Taipei City, Taiwan), or anti-COX IV rabbit polyclonal antibody (ab16056, Abcam plc) in fresh blocking solution overnight at 4°C. After washing with wash solution (Kirkegaard and Perry Laboratories), the cells were incubated with 1 μg/mL Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA) or Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probes) for 1 h at room temperature. After washing, the specimens were mounted using VECTASHIELD mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were obtained using a Confocal Laser Scanning Biological Microscope (FV10i-DOC, Olympus, Tokyo, Japan).
4
Cellular Transduction of TAT-Conjugated Proteins
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The transduction of TAT-conjugated IAPs into IEC6 cells was measured with immunofluorescent staining, for which cells were cultured in 35-mm glass-base dishes (IWAKI, Chiba, Japan). Following exposure to TAT-IAPs for 1 h, cells were fixed with 4% paraformaldehyde for 30 min, permealized with 0.5% Triton X-100 for 5 min, and blocked for 30 min in PBS containing 10% normal goat serum and 1% BSA for 30 min at room temperature. Cells were incubated for 2 h at room temperature with Anti-HA (1:200 dilution; 3F10; Roche Applied Science), and then for 1 h at room temperature with Alexa Fluor 488-goat anti-rat IgG (1:600 dilution; Life Technologies). The solution of secondary antibody also contained 5 μg/ml of Hoechst 33258 to counterstain nuclear DNA. All antibodies were diluted with PBS containing 1% BSA. Dishes were mounted in drops of Vectashield (Vector Laboratories, Burlingame, CA). Fluorescence microscopy was performed using an Olympus IX70 instrument and Meta Morph software (Molecular Devices, Sunnyvale, CA).
For the fluorescence microscopy of GFP-TAT and GFP, IEC6 cells were exposed to 2.5 μg/ml of the proteins for 1 h. After the growth medium was replaced by Hanks' Balanced Salt Solution (HBSS; Life Technologies), the cells were examined with the IX70 instrument.
For the fluorescence microscopy of GFP-TAT and GFP, IEC6 cells were exposed to 2.5 μg/ml of the proteins for 1 h. After the growth medium was replaced by Hanks' Balanced Salt Solution (HBSS; Life Technologies), the cells were examined with the IX70 instrument.
5
Cellular Uptake of siRNA-Loaded LNPs
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SK-OV-3-luc cells were seeded (1.0 × 105 cells/well) on 35 mm glass base dishes (Iwaki, Tokyo, Japan) and cultured in McCoy’s 5A (Modified) medium containing 10% fetal bovine serum (FBS). The cell medium was then replaced with new Optimem containing Alexa Flour 647-siGL3 encapsulating LNPs. After incubation for 72 h, the nuclei and lysosomes were stained with Hoechst 33,342 (Dojindo, Kumamoto, Japan) and LysoTracker Red DND-99 (Thermo Fisher Scientific, Inc. Waltham, MA, U.S.A.), respectively. LSM710 (Carl Zeiss, Oberkochen, Germany) equipped with a Plan-Apochromat 40× oil immersion objective lens (aperture: 1.4) was used for each confocal laser scanning microscopy (CLSM) measurement. The excitation wavelengths were 405 nm for Hoechst 33,342, 488 nm for LysoTracker Red DND-99, and 633 nm for Alexa Flour 647-siGL3. For quantitative analysis of cellular uptake study, the regions-of-interest (ROIs) were selected manually to identify each single cell (n = 30). The fluorescence signals (633 nm) detected from ROIs were quantified using ZEN Microscopy software (ver. 6.2.0.500).
6
Apoptosis Pathway Analysis in HeLa Cells
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For active Caspase-3 staining, HeLa cells were cultured in 35 mm glass base dishes (Iwaki) and, when indicated, transfected with either empty pBI-CMEF, pBI-CMEF-FLAG-Bcl2l10, pBI-CMEF-FLAG-IRBITS68A or pBI-CMEF-FLAG-IRBITS68A-FLAG-Bcl2l10. 24 hr after transfection, cells were treated with DMSO (1/1000, Sigma-Aldrich) for 24 hr, 1 µM staurosporine (LKT Laboratories) for 4 hr, 2 µM thapsigargin (Calbiochem) for 24 hr or 20 µM tunicamycin (Sigma-Aldrich) for 24 hr and then stained using the Image-iT LIVE Green Caspase-3 and −7 Detection Kit (ThermoFisher Scientific) according to the manufacturer's instructions. Images were acquired with a fluorescence microscope (Biozero BZ-8100, Keyence).
For western blot analysis of apoptosis, cells were transfected, when indicated, with empty pHM6 or pHM6-IRBIT and treated, 24 hr after transfection, with DMSO (1/1000, Sigma-Aldrich) for 24 hr, 1 µM staurosporine (LKT Laboratories) for 4 hr, 2 µM thapsigargin (Calbiochem) for 24 hr or 20 µM tunicamycin (Sigma-Aldrich) for 24 hr. After treatment, cells were lysed in TNE buffer, the protein concentration of each sample was determined using the Bradford assay (Bio-Rad) and equivalent amounts of protein (10 μg) were analyzed by immunoblotting with appropriate antibodies.
For western blot analysis of apoptosis, cells were transfected, when indicated, with empty pHM6 or pHM6-IRBIT and treated, 24 hr after transfection, with DMSO (1/1000, Sigma-Aldrich) for 24 hr, 1 µM staurosporine (LKT Laboratories) for 4 hr, 2 µM thapsigargin (Calbiochem) for 24 hr or 20 µM tunicamycin (Sigma-Aldrich) for 24 hr. After treatment, cells were lysed in TNE buffer, the protein concentration of each sample was determined using the Bradford assay (Bio-Rad) and equivalent amounts of protein (10 μg) were analyzed by immunoblotting with appropriate antibodies.
7
Visualizing Tight Junction Proteins in Cells
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The cells were grown on 35‐mm glass‐base dishes (Iwaki, Chiba, Japan) coated with rat tail collagen. They were fixed with cold acetone and ethanol (1:1) at 20°C for 10 min. After rinsing in PBS, the sections and cells were incubated with polyclonal anti‐claudin‐1 (1:100) and monoclonal anti‐claudin‐4 (1:100) antibodies, and polyclonal anti‐claudin‐4 (1:100), and monoclonal anti‐his‐tag (1:1000) antibodies at room temperature for 1 h, and then with Alexa Fluor 488 (green)‐conjugated anti‐rabbit IgG (1:200) and Alexa Fluor 594 (red)‐conjugated anti‐mouse IgG (1:200) at room temperature for 1 h. DAPI (Sigma‐Aldrich) was used for counterstaining of nuclei in the cells. The specimens were examined, using a confocal laser scanning microscope (LSM510; Carl Zeiss, Jena, Germany).
8
Imaging Mitotic Spindle Formation
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HeLa-S3 cells inducibly expressing 6Myc-INCENP-WT and -S894A were plated in 35 mm glass-base dishes (IWAKI, Tokyo, Japan) and treated with 10 ng/ml nocodazole for 18 h in the presence of 2 μg/ml doxycycline (Dox). After washing, cells were maintained in Dox-containing medium at 37 °C using a stage heater on a model IX71 fluorescence microscope (Olympus) equipped with a humidity chamber and CO2 injection control system. The images were obtained using a high-sensitivity CoolSNAP-HQ CCD camera (Olympus), a Z-axis motor, and a 40x UplanApo Oil Iris objective lens, and were collected at 2 min intervals using the MetaMorph imaging analysis software (Universal Imaging Ltd., Buckinghamshire, UK).
9
Visualizing Exosome Cellular Uptake
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To observe the cellular uptake of exosomes, they were labeled with PKH26 (Sigma-Aldrich, St. Louis, MO, USA). A mixture of exosomes and PKH26 solution was incubated for 5 min at room temperature and then put in a Vivacon 500/100k filter unit (Sartorius, Gottingen, Germany) followed by centrifugation at 14,000 × g for 2 min at room temperature. The pellet containing PKH26-labeled exosomes was washed with PBS four times. PKH26-labeled exosomes were added into medium in which Hoechst 33342 (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA)-labeled lymphoma cells were cultured, and then incubated on 35-mm glass base dishes (Iwaki, Shizuoka, Japan) for 12 h at 37 °C. Cellular uptake of exosomes was observed with a confocal fluorescence microscope (TiE-A1R, Nikon, Tokyo, Japan).
10
Zebrafish Embryo Imaging and Manipulation
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For fixation and live imaging, embryos were anaesthetized with 0.01% tricaine. For live imaging, embryos were embedded in 0.8% low melting temperature agarose (Type IV-A, Sigma, USA) in 35mm glass-base dishes (Iwaki, Japan) at 28°C. Detailed procedures for the standard embryological experiments including, dechorionation, fixation, in situ hybridization, immunohistochemistry, microinjection and cell transplantation were carried out according to previously published procedures31 (link). Cells were transplanted to the region fated to become the eye and Cuvier's duct according to our fate map33 (link).
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