Retinal dissection was performed following established protocols in the Frankfort lab (Frankfort et al., 2013 (link); Tao et al., 2020b (link)). Dissected whole-mount retinas were fixed with 4% paraformaldehyde for 1 h at room temperature and blocked with 10% donkey serum overnight. Retinas were then incubated in primary antibodies [Collagen IV (EMD Millipore cat#AB756p; 1:300), CD31 (BD bioscience cat#550274; 1:50), and RBPMS (Phospho solutions cat#1832; 1:250)] diluted with 3% donkey serum for 5 days at 4°C, followed by overnight incubation at 4°C in secondary antibodies [Alexa fluor 647 donkey anti-rabbit (Jackson Immuno Research Labs cat# 711-605-152; 1:300), Cy3 donkey anti-rat (Jackson Immuno Research Labs cat#712-165-153; 1:300), Alexa fluor 488 donkey anti-guinea pig (Jackson Immuno Research Labs cat#706-545-148; 1:300), and Hoechst 33,342 nuclear staining (Invitrogen cat#H3570; 1:1,000)] diluted with 3% donkey serum.
Hoechst 33 342 nuclear staining
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hoechst 33,342 is a fluorescent dye used for nuclear staining in biological applications. It binds to DNA and emits a blue fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Rbpms, by PhosphoSolutions (1 mentions)
Alexa fluor 488 donkey anti guinea pig, by Jackson ImmunoResearch (1 mentions)
Donkey anti rabbit alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Donkey anti rat cy3, by Jackson ImmunoResearch (1 mentions)
Collagen 4, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
Anti yap1, by Cell Signaling Technology (1 mentions)
Tirf microscope, by Nikon (1 mentions)
Anti cd98, by BioLegend (1 mentions)
Anti clathrin heavy chain, by Abcam (1 mentions)
Anti ap2, by Abcam (1 mentions)
Coverslip chambers, by Thermo Fisher Scientific (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Anti glut1, by Abcam (1 mentions)
4 protocols using hoechst 33 342 nuclear staining
1
Retinal Dissection and Immunostaining
2
Immunofluorescence Analysis of Esophageal Biopsies
3
Hoechst Nuclear Staining for Cytotoxicity Analysis
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Hoechst 33,342 nuclear staining (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to detect the nuclear changes. In total, 5000 cells/well were seeded in a 96-well black plate for 24 h at 37 °C and 5% CO2. After this period, EcTI (25, 50, and 100 µM) was added to each well and incubated for another 24 h, washed thoroughly with PBS, and incubated for 10 min with Hoechst staining solution (2 µg/mL, diluted in PBS, 100 µL/well). Nuclear staining was observed with an inverted fluorescence microscope (Leica) using a 10× objective lens and a set of blue filter cubes (excitation: 394–410 and emission: 435–485). The nuclear area (µm2) was measured using ImageJ software, using the Make Binary Tool and Measure. The percentages of the nuclear area were calculated using the control group compared to the EcTI treatment groups [39 (link)].
4
Immunocytochemical Analysis of SUM-159 Cells
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SUM-159 cells plated on coverslip chambers (Thermo Fisher, Cat. # 155379) were fused at their corresponding time points, fixed with 4% PFA (EMS), and then permeabilized and blocked with blocking solution (0.5% Triton X-100, 10% BSA in PBS) for 1 h. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C. After 3 washes (10 min) with PBS, cells were incubated with Alexa Fluor-conjugated secondary antibodies. The following primary antibodies were used: Anti-Yap1 (Cell Signaling 14074S, 1:250), Anti-p21 (Cell Signaling, Cat. # 2947S, 1:200), Anti-pH3 (anti-Phospho-Histone H3 (Ser10); Cell Signaling, Cat. # 3377, 1:200), Anti-clathrin heavy chain (Abcam, Cat. # ab21679, 1:200), Anti-AP-2 (Abcam, Cat. # ab189995, 1:200), Anti-Glut1 (Abcam, Cat. # ab40084, 1:100), Anti-CD98 (BioLegend, Cat. # 315602, 1:200) and Anti-CD147 (BioLegend, Cat. # 306202, 1:200). For imaging and quantification, at least a total of 15 fields of view were randomly chosen by Hoechst 33342 nuclear staining (Thermo Fisher, Cat. # 62249) and imaged by Zeiss LSM880 confocal or NIKON TIRF microscope. At least three different samples were quantified per treatment type at each respective time point.
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