In a 12-well plate, HEK293T cells at 40% to 50% confluence were transfected with the indicated plasmids (1,000 ng) for 24 h, then cells were washed twice with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and fixed in 4% paraformaldehyde at room temperature for 10 min. Following permeabilization with 0.1% Triton X-100 buffer for 5 min, cells were washed three times with PBS containing 0.1% BSA, then blocked with PBS containing 5% BSA for 1 h. The cells were incubated with the primary antibody overnight at 4°C, followed by incubation with DyLight 488-conjugated anti-rabbit IgG and DyLight 568-conjugated anti-mouse IgG for 1 h. After three washes with PBS (containing 0.1% BSA), the cells were counterstained with Hoechst-33258 solution (Invitrogen, Waltham, MA, USA) for 5 min and then washed three more times with PBS. Finally, the cells were analyzed using a confocal laser scanning microscope (Leica TCS SP8 STED, Leica, Germany). The antibodies used are listed in Table 3 .
Hoechst 33258 solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hoechst 33258 solution is a fluorescent dye commonly used in molecular biology applications. It binds to the minor groove of double-stranded DNA and emits blue fluorescence upon excitation. The solution can be used to detect and quantify DNA in various assays.
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Lab products found in correlation
Leica tcs sp8 sted, by Leica camera (2 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Fitc conjugated, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Forma 3111, by Thermo Fisher Scientific (1 mentions)
Penicillin a, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
5 protocols using hoechst 33258 solution
1
Immunofluorescence Staining of HEK293T Cells
2
Immunofluorescence Staining of HEK293T Cells
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In a 12-well plate, 40% to 50% HEK293T cells were transfected with the indicated plasmids (1000 ng) for 24 h; then, cells were washed twice with phosphate-buffered saline (PBS) (containing 0.1% bovine serum albumin [BSA]) and fixed in 4% paraformaldehyde at room temperature for 10 min. Following permeabilization with 0.1% Triton X-100 buffer for 5 min, cells were washed three times with PBS (containing 0.1% BSA) and blocked with PBS containing 5% BSA for 1 h. The cells were then incubated with the primary antibody overnight at 4°C, followed by incubation with DyLight 488-conjugated anti-rabbit IgG and DyLight 568-conjugated anti-mouse IgG for 1 h. After three times washing with PBS (containing 0.1% BSA), cells were counterstained with Hoechst 33258 solution (catalog no. H1398; Invitrogen, San Diego, CA, USA) for 5 min and then washed three more times with PBS. Finally, the cells were analyzed using a confocal laser scanning microscope (Leica TCS SP8 STED [STimulated Emission Depletion]; Leica, Germany). The antibodies used are listed in Table 1 .
3
Riboflavin-Induced Oxidative Stress in Bat Fibroblasts
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Primary bat fibroblasts were grown in biological triplicates to 50% confluency in 24 well plates (Nunc), incubated with riboflavin (2 h; 200, 100, 50, 25, 12.5 and 0 μg ml−1 concentrations), then illuminated in the presence of ROS sensor CellROX Green Reagent (1 μM; Invitrogen) in a botanical climabox (30 min, light intensity 110 μmol of photons m−2 s−1). Cells were detached from cultivation wells with trypsin (0.1%, 200 μl per well, Sigma-Aldrich, USA). trypsinization was stopped with complete cultivation medium (200 μl) and Hoechst 33258 solution was added (1 μM, 10 min; Invitrogen). Intensity of CellROX Green Reagent fluorescence was quantified in cell population gated as viable singlets by fluorescence-activated cell sorter LSR II (Becton Dickinson, USA).
4
Bacterial FISH and Lectin Staining in Colon
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FISH was performed as previously described by Molloy et al.110 (link) Colon fragments were fixed in methacarn for 3 h at 4°C and 5 µm coronal slices were obtained. Sections were incubated with 100 nM bacterial probes (GCTGCCTCCCGTAGGAGT; FITC-conjugated; Sigma), 20 mg/mL lectin-Ulex europaeus agglutinin-I (UEA-I; Tetramethyl-rhodamine-conjugated, Sigma), 10 mg/mL Hoechst 33,258 solution, and mounted with SlowFade Gold medium (Thermo Fisher Scientific). Images were acquired using a Zeiss LSM-780 confocal microscope (Carl Zeiss, Oberkochen, Germany). Samples were imaged with a 63×/1.4NA oil-immersion objective at 3× with a 1024 3 1024 frame size.
5
Isolation and Culture of Dermal Papilla Cells
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Primary DPCs were isolated from the follicle bulbs of C57BL/6N mice whiskers as previously described [81 (link),82 (link)]. The isolated DPCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Co., Carlsbad, CA, USA) with 100 units/mL each of penicillin A and streptomycin (Gibco BRL, Grand Island, NY, USA), and 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL Grand Island, NY, USA). Cells were grown at 37 °C in fully humidified 5% CO2 (Forma 3111, Thermo Fisher Scientific, Waltham, MA, USA).
The primary DPCs (5 × 103 or 3 × 103 cells/well) were seeded onto 24-well plates. PGA-4HGF (2% (v/v)) (4HGF:PGA:chitosan = 1:1:2, 1:1:4, or 1:1:8), PGA-control (PGA:chitosan = 1:2, 1:4, or 1:8), and 4HGF were treated to DPCs, and images of DPC proliferation (40× magnification) were taken after 96 h using a CCD camera (Point Grey Research Inc., Richmond, BC, Canada) and analyzed using the MetaMorph software (Universal Imaging, West Chester, PA, USA).
To quantify the number of DPCs, 1:1:4 PGA-4HGF (2% (v/v)), 1:4 PGA-control, and 4HGF were treated to DPCs with Hoechst 33258 solution (Thermo Fisher Scientific, Waltham, MA, USA). The image of stained DPCs (100× magnification) was taken after 24 h (Nikon Eclipse Ti, Nikon Instruments Inc., Kobe, Japan) and then was analyzed using the Image J software (Wright Cell Imaging Facility, version, city, if any state, country).
The primary DPCs (5 × 103 or 3 × 103 cells/well) were seeded onto 24-well plates. PGA-4HGF (2% (v/v)) (4HGF:PGA:chitosan = 1:1:2, 1:1:4, or 1:1:8), PGA-control (PGA:chitosan = 1:2, 1:4, or 1:8), and 4HGF were treated to DPCs, and images of DPC proliferation (40× magnification) were taken after 96 h using a CCD camera (Point Grey Research Inc., Richmond, BC, Canada) and analyzed using the MetaMorph software (Universal Imaging, West Chester, PA, USA).
To quantify the number of DPCs, 1:1:4 PGA-4HGF (2% (v/v)), 1:4 PGA-control, and 4HGF were treated to DPCs with Hoechst 33258 solution (Thermo Fisher Scientific, Waltham, MA, USA). The image of stained DPCs (100× magnification) was taken after 24 h (Nikon Eclipse Ti, Nikon Instruments Inc., Kobe, Japan) and then was analyzed using the Image J software (Wright Cell Imaging Facility, version, city, if any state, country).
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