Immunofluorescence staining was performed as we previously described8 (link). Briefly, after washing twice with PBS, HepG2 cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, at indicated times. Then, cells permeabilization with 0,1% Triton X-100 twice for 10 min, and blocking with 2% BSA were performed. Cells were incubated overnight with primary antibodies, including rabbit anti-p21 (1:100, Santa Cruz), rabbit anti-Ki-67 (1:100 Millipore) and mouse anti-Mdm-2 (1:100, Santa Cruz). Secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100, Thermo Fisher Scientific) were used according to the manufacturer’s instructions. The samples were incubated at room temperature for 1 h and washed 3 times with PBS. Nuclei were counterstained with DAPI, and samples were mounting with prolong Gold antifading solution (Thermoscientific). Primary antibody was omitted in negative controls (data not shown). Cells were visualized and photomicrographed under an inverted fluorescence microscope (Nikon). Positive Alexa 488 cells were analyzed and quantified using FIJI-Image J software (Bethesda, MD, USA).
Antifading prolong gold solution
Manufactured by Thermo Fisher Scientific
ProLong Gold Antifade Mountant is a ready-to-use aqueous mounting medium designed to reduce photobleaching of fluorophores. It contains an antifadent and preservatives to extend the fluorescent signal of labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Inverted fluorescence microscope, by Nikon (3 mentions)
Hrp conjugated secondary antibody, by Roche (3 mentions)
Double dig labeled mercury lna microrna probe, by Qiagen (2 mentions)
Discovery ultra, by Roche (2 mentions)
Polylysine d coated coverslips, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Mouse anti mdm2, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p21, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti ki67, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti albumin, by Santa Cruz Biotechnology (1 mentions)
Anti ki67, by Merck Group (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti p21, by Santa Cruz Biotechnology (1 mentions)
Cleaved parp, by Thermo Fisher Scientific (1 mentions)
Formalin, by Thermo Fisher Scientific (1 mentions)
U cmad3, by Olympus (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Anti dig antibody, by Merck Group (1 mentions)
8 protocols using antifading prolong gold solution
1
Immunofluorescence Staining of Cell Markers
2
Immunocytochemical Characterization of Differentiated Cells
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At indicated differentiation times, culture cells were washed with PBS twice and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Then, cells were permeabilized with 0.1% Triton-X twice for 10 min, blocked with 2% BSA and incubated overnight with primary antibodies, including anti-SSEA-4 (mouse, 1:100, Cell Signaling), anti-albumin (mouse, 1:100, Santa Cruz Biotechnology), anti-Ki-67 (rabbit, 1:100, Millipore), anti-p21 (rabbit, 1:100, Santa Cruz Biotechnology) and anti-P-ERK (rabbit, 1:100, Cell Signaling). No permeabilization was performed for SSEA-4 detection. Secondary antibodies including FITC-conjugated anti-mouse IgG (goat, 1:100, Sigma) and Alexa Fluor 488-conjugated anti-rabbit IgG (goat, 1:100, Thermo Fisher Scientific) were used according to the manufacturer’s instructions. The samples were incubated at room temperature for 1 h. After further washings with PBS and counterstained with DAPI, samples were mounting with prolong Gold antifading solution (Thermoscientific). In negative controls, primary antibody was omitted for each respective fluorescent tracer (data not shown). Cells were visualized and photomicrographed under an inverted fluorescence microscope (Nikon). Positive FITC or Alexa 488 cells were analyzed and quantified using FIJI-Image J software (Bethesda, MD, USA)
3
Immunofluorescent Localization of ER and RelA
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Bewo cells were grown on coverslips in DMEM-F12 10% FBS. The next day they were transiently transfected with Rel A and/or HEGO expression plasmids and were treated with 100 nM estradiol at the times indicated in the figures. Then they were washed three times with PBS, fixed 20 min with 3% p-formaldehyde and permeabilized with 0.1% Triton X-100 as described (Quinta et al. 2010) (link). After the fixation, the cells were washed three times with PBS, and blocked for 30 min with AFI solution (BSA 0.5%, Tween 20 0.2%, sodium azide 0.1%, Tris 50 nM pH 8). The primary antibodies were diluted in AFI solution and were added to the cells ON at 4°C: rabbit polyclonal anti-ERα (1:50, Santa Cruz Biotechnology) or mouse monoclonal anti-Rel A (1:100 Santa Cruz Biotechnology). This incubation was followed by three washes with PBS for 10 min and then the cells were incubated with the secondary antibodies for 1 h: Alexa 488 nm anti rabbit IgG (1:100, Thermo Fisher) or Alexa 563 nm anti mouse IgG (1:100, Thermo Fisher). After further washings with PBS and counterstained with DAPI, samples were mounting with prolong Gold antifading solution (Thermo Scientific). The images were taken using an inverted fluorescence microscope (Nikon) and analyzed using the software Image J version 1.45 (NIH).
4
In Situ Hybridization of microRNA in FFPE Tissue
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Deparrafinization and rehydration of the formalin-fixed, paraffin-embedded tissue sections used xylene and an ethanol dilution series. Sections of tissue sections were digested with 15 µg/mL proteinase K for 20 minutes at room temperature and loaded onto Ventana Discovery Ultra for in situ hybridization analysis. Tissue slides were incubated with double-DIG labeled mercury LNA microRNA probe (exiqon) for two hours at 55°C. Three percent H2O2 was used to inactivate endogenous peroxidases. Following polyclonal anti-DIG antibody and HRP-conjugated secondary antibody (Ventana) incubation, tyramine-conjugated fluorochrome (TSA) reaction was performed for twelve minutes. Sequential TSA rounds were performed for the detection of proteins using the same protocol. Slides were mounted with antifading ProLong Gold Solution (Life Technologies).
5
Immunofluorescence Analysis of NF-κB Pathway
6
In Situ Hybridization for miRNA and lncRNA Detection
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The formalin-fixed paraffin embedded tissue sections were dewaxed in xylenes and rehydrated through an ethanol dilution series60 . Tissue sections were digested with 5 μg ml−1 proteinase K for 15 min at RT, and were then loaded onto Ventana Discovery Ultra for in situ hybridization analysis. The tissue slides were incubated with double-DIG labeled mercury LNA probe (Exiqon) for 2 h. The slides were treated with 3% H2O2 to inactivate endogenous peroxidase. Followed by a rabbit polyclonal anti-DIG antibody (Sigma, Cat.#: D7782, 1:10,000 dilution) and HRP conjugated secondary antibody (Ventana, Cat.#: 760–4311, ready to use) incubation, tyramine-conjugated fluorochrome (TSA) reaction was performed for 12 min. Finally, the slides were mounted with antifading ProLong Gold Solution (Life Technologies). For the chromogenic detection of miRNAs, miRNA signal was recognized by anti-DIG antibody, and alkaline phosphatase (AP) conjugated second antibody (Ventana, Cat.#: 760–4314, ready to use) using NBT-BCIP as the substrate. The following probes were used: CCNE2: 5′-TGCTCTTCGGTGGTGTCATAA-3’ and uc.339: 5′-AGATGGAGGATCGGTGTGAAA-3′. For U6 control probe # 99002-15 (Exiqon) was used. For miR-339-3p, probe # 38578-15 (Exiqon) and for miR-663b-3p, probe # 21359-15 (Exiqon) were used. The uc339 signal in ISH was amplified by anti-rab-HRP antibody and Tyramide signal amplification (TSA) system.
7
Quantifying DNA Damage Markers
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3 × 105 cells were adhered onto polylysine D -coated coverslips (Sigma-Aldrich) during a 45-min incubation at 37 °C, fixed in 10% formalin (Fisher Scientific, Pittsburgh, PA, USA) and permeabilized in 1% Triton-X 100 in PBS. Coverslips were blocked for 30 min in 5% bovine serum albumin (Sigma-Aldrich) in PBS with 0.1% Tween-20, probed with γH2AX (S139), phospho-RPA32 (S4/S8) or Ki-67 antibodies (conjugated with AlexaFluor647; Life Technologies) and then with DyLight 488 goat anti-rabbit antibodies (Thermo Scientific, Carlsbad, CA, USA). Coverslips were mounted with anti-fading ProLong Gold Solution (Life Technologies) with 4′,6-diamidino-2-phenylindole (DAPI, for nuclear counterstaining). Fluorescent images were captured with a Zeiss AxioCam MRm camera mounted on a Zeiss Observer.Z1 microscope (Zeiss, Jena, Germany). Cells positive for γH2AX or phospho-RPA were counted in six high-power fields per condition ( × 20 magnification) and referenced to the total number of cells quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
In Situ Hybridization of microRNA in FFPE Tissue
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