5-μm frozen section from each sample was fixed in acetone at 4 °C overnight. After washing, sections were blocked with 1 % bovine serum albumin for 30 min and incubated overnight at 4 °C with rabbit anti-human CCNG1 antibody (1:50). Then sections were incubated with anti-rabbit IgG–FITC (1:100, Santa Cruz Biotechnology) for 2 h at room temperature in the dark. Nuclei were stained with diaminophenylindole (1 μg/mL; Sigma-Aldrich) for 5 min at room temperature. Coverslips were mounted and imaged using a laser confocal microscope (Olympus, Tokyo, Japan).
Diaminophenylindole
Manufactured by Merck Group
Sourced in United States
Diaminophenylindole is a fluorescent dye used in laboratory applications for the detection and visualization of nucleic acids, such as DNA and RNA. It binds to the minor groove of double-stranded DNA, emitting a blue fluorescence when excited by ultraviolet light. This dye is commonly used in various techniques, including fluorescence microscopy, flow cytometry, and gel electrophoresis, to stain and detect nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Laser confocal microscope, by Olympus (2 mentions)
Anti rabbit igg fitc, by Santa Cruz Biotechnology (1 mentions)
Chicken polyclonal to ibdv, by Abcam (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Abmart (1 mentions)
Anti vimentin, by BD (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
5 protocols using diaminophenylindole
1
Immunofluorescent Visualization of CCNG1
2
IBDV Immunohistochemistry in Tissue Sections
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For IHC-fluorescent, tissue sections on poly-L-Lysine coated slides were deparaffinized and rehydrated, and antigen retrieval was done in citrate buffer solution. Blocking of non-specific protein binding and endogenous peroxide was followed by overnight incubation in primary antibody (Chicken polyclonal to IBDV, Abcam, UK). It was followed by incubation with fluorescein isothiocyanate-conjugated goat polyclonal secondary antibody to chicken IgY-Fc (Merck, India) for 20 min in the dark. Counterstaining was done using diamino phenylindole (5 mg/ml, Sigma, USA) for 10 min and after washing mounted in an aqueous glycerol mounting media. Omission of primary antibodies was used for negative control. The slides were viewed under fluorescent microscope (Nikon eclipse).
The antibody was standardized at the dilution of 1:200 for the above two techniques.
The antibody was standardized at the dilution of 1:200 for the above two techniques.
3
Lamellipodia Visualization in Fixed Cells
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Cells were grown on glass coverslips, fixed with PBS containing 4% formaldehyde for 15 minutes, and permeabilized with 0.2% Triton X-100 in PBS for 15 minutes at room temperature. After washing with PBS, the cells were incubated overnight at 4°C with Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) to visualize the lamellipodia. Nuclei were stained with 1 mg/mL diaminophenylindole (Sigma-Aldrich Co.) for 5 minutes at room temperature. The coverslips were then mounted with SlowFade Gold antifade reagent (Thermo Fisher Scientific) and observed under a confocal laser microscope (Olympus Corporation, Tokyo, Japan).
4
Immunofluorescence Analysis of 3D Tissue Models
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The 3D rBM gels, as well as tissue sections, were prepared by mixing cultures with fresh collagen following embedment and freezing in sucrose with Tissue-Tek OCT compound (Miles Laboratories), then sectioning them in 10- to 20-µm-thick slices for analysis. All samples were incubated with primary mAbs followed directly by either FITC-, Texas red-, or Alexa Fluor–conjugated secondary antibodies and DAPI, when indicated. Nuclei were counterstained with diaminophenyl-indole (Sigma-Aldrich). Images were compared and quantified based on fluorescence intensity signal following minimal thresholding to subtract background. For mouse studies, when mice were killed, lesions were photographed, dissected, measured, macroscopically analyzed, fixed in 4% paraformaldehyde (PFA), and paraffin embedded. H&E sections were evaluated for histopathological evidence of tumor phenotype, and tissue sections were analyzed by immunofluorescence as described.
5
WNT2 Regulation in Cervical Cancer
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The SiHa cervical cancer cell line was obtained from the Shanghai Institutes for Biological Sciences Cell Bank (Shanghai, China), and was tested and authenticated by short tandem repeat genotyping. The cells were cultured in Roswell Park Memorial Institute 1640 medium (GIBCO BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA). Three human short hairpin RNA (shRNA) sequences for repressing WNT2 expression were cloned; S1 Table lists their sequences. Retroviral production and infection were performed as described previously[16 (link)]. Mouse anti—E-cadherin, anti-vimentin (BD Transduction Laboratories, Lexington, UK), and anti–β-tubulin (Abmart, Shanghai, China), and rabbit anti-WNT2 (Abcam, Cambridge, UK) and anti–β-catenin (Cell Signaling Technology, Danvers, MA, USA) antibodies were used in this study. Rhodamine-conjugated antibodies (Invitrogen, Carlsbad, CA, USA) and diaminophenylindole (Sigma-Aldrich, St Louis, MO, USA) were used in immunofluorescence analysis.
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