Adipogenic staining was performed as described previously (Eom et al., 2018 (link)). In brief cells were plated in a 96-well plate in 200 μl ASC media (DMEM/F12 media (Life Technologies) supplemented with 10% FBS, 1% GlutaMax (Life Technologies, Auckland) and 1% penicillin/streptomycin (Life Technologies) and cultured at 37°C, 5% CO2. On day four, 100 μl of media was replaced with adipogenic differentiation media (ASC media with 1 µM dexamethasome, 10 µM insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 200 µM indomethacin (all from Sigma Aldrich, Auckland) and standard ASC media was added to control wells. Half media changes were performed every 3 days until day 14. Cells were then subjected to immunocytochemistry using a 1:200 dilution of rabbit antihuman FABP4 polyclonal antibody (Cat #10004944, Cayman Chemicals) and then incubated with a 1:200 dilution Alexa Fluor® 488 conjugated goat antirabbit IgG secondary antibody (Cat # A11008, Molecular Probes®) and 1:2,000 diluted DAPI (Cat# D3571, Molecular Probes®). Fluorescent images were taken using the ImageXpress Micro XLS high content screening system (Molecular Devices™). Nine images were taken per well at 10 × magnification and quantitative data was generated using the MetaXpress v 5.3.0.1 (Molecular Devices™) software.
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody is a fluorescently labeled antibody that binds to rabbit immunoglobulin G (IgG) molecules. It is designed for use in immunoassays and other applications requiring the detection of rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Axio imager a1 inverted fluorescence microscope, by Zeiss (3 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (3 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (2 mentions)
Anti histone h3 antibody, by Abcam (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Alexa fluor 555 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Eclipse ni e, by Nikon (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Imagexpress micro xls high content screening system, by Molecular Devices (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Fv10 asw, by Olympus (1 mentions)
Rabbit anti caspase 3 antibody, by Abcam (1 mentions)
42 protocols using alexa fluor 488 conjugated goat anti rabbit igg secondary antibody
1
Adipogenic Differentiation Quantification
2
Lipid Raft and Ret Patching Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipid raft patching was induced as described by Fra et al. and Peter et al. with slight modifications [29 (link),30 (link)]. Briefly, for all fluorescence microscopy experiments, the cells were attached to 35 mm poly-l -lysine-coated dishes at a density of 2 × 105. Cholera toxin subunit B (CT-B) labeled with Alexa Fluor 594 (Molecular Probes Inc., Eugene, OR, USA) was used at 10 mg/mL in PBS to label ganglioside GM1, which selectively partitions into lipid rafts. An anti-CT-B antibody (1:200 in PBS; Molecular Probes Inc.) was used to crosslink the CT-B-labeled lipid rafts into distinct patches on the plasma membrane. The cells were then incubated for 10 min and 15 min at 4 °C. The presence of lipid raft aggregation or patching was confirmed after CT-B and anti-CT-B labeling. For fixation, the cells were directly treated with 4% paraformaldehyde in PBS for 15 min.
Ret patching was induced in the same manner. The cells were incubated with a primary anti-Ret antibody (1:1000, Abcam) overnight at 4 °C, and this was followed by incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes Inc.) for 2 h at room temperature. Confocal microscopy (FV10-ASW, Olympus, Tokyo, Japan) was performed with a 60× oil immersion objective, using laser excitation at 350, 488, and 594 nm.
Ret patching was induced in the same manner. The cells were incubated with a primary anti-Ret antibody (1:1000, Abcam) overnight at 4 °C, and this was followed by incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes Inc.) for 2 h at room temperature. Confocal microscopy (FV10-ASW, Olympus, Tokyo, Japan) was performed with a 60× oil immersion objective, using laser excitation at 350, 488, and 594 nm.
3
Immunofluorescent Localization of TRPM8 in BEAS-2B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEAS2B cells were cultured on 22-mm round coverslips (BD Biosciences, Bedford, MA, USA) until 75% confluence and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes. Cells were washed 3 times with PBS, and nonspecific binding was blocked using a solution of 5% bovine serum albumin in PBS. The cells were rinsed 3 times with PBS and incubated at room temperature (RT) for 2 hours with a rabbit polyclonal immunoglobulin G (IgG) antibody fraction specific for human TRPM8 (Abcam, Cambridge, MA, USA), diluted 1:250 in the blocking solution. The cells were washed and treated for 1 hour at RT with an Alexa-Fluor 488 conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR, USA) at a dilution of 1:500 in the blocking solution. The nuclei were counterstained blue using 4′,6-diamidino-2-phenylindole (DAPI) at 300 nM dilution in PBS. Controls consisted of untreated cells or cells treated with either primary or secondary antibodies alone. Images were collected using a Zeiss LSM700 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with filters to visualize green fluorescent protein and DAPI. Immunoreactivity of TRPM8 was detected as green fluorescence.
4
Immunofluorescent Detection of NFATc1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells cultured on glass coverslips were washed with phosphate-buffered saline (PBS), fixed using 4% paraformaldehyde in PBS for 10 min, and then permeabilized with 0.05% Triton-X 100 in PBS for 5 min. Fixed cells were incubated with rabbit anti-NFATc1 antibody (Gene Tex, Inc., Irvine, CA; dilution, 1:200 in PBS) at 4°C overnight after blocking of the nonspecific binding sites with 5% normal goat serum in PBS for 30 min at room temperature. After washing with PBS, the antibody was visualized by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1 μg/mL; Molecular Probes, Eugene, OR) for 30 min at room temperature. Nuclear staining was performed using 4,6-diamidino-2-phenylindole (DAPI) dye (Dojindo, Kumamoto, Japan).
5
TRPM8 Immunofluorescence in BEAS-2B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEAS-2B cells were cultured on a 22-mm round cover slip (BD Biosciences, Bedford, MA, USA) until 75% confluence and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. The cells were then washed three times with PBS, and nonspecific binding was blocked using a solution of 5% bovine serum albumin in PBS. The cells were rinsed three times with PBS and incubated at room temperature for 2 h with a rabbit polyclonal IgG antibody fraction specific to human TRPM8 (Abcam), which was diluted 1:250 in the blocking solution. The cells were washed and treated for 1 h at room temperature with an Alexa-Fluor 488 conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR, USA) at a dilution of 1:500 in the blocking solution. The nuclei were counterstained blue using 4′, 6-diamidino-2-phenylindole (DAPI) at 300 nM dilution in PBS. Controls consisted of untreated cells or cells treated with either primary or secondary antibodies alone. Images were collected using a ZEISS LSM700 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with filters to visualize green fluorescent protein and DAPI. The immunoreactivity of TRPM8 was detected as green fluorescence.
6
Immunofluorescence Staining of Retinal Caspase-3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was performed as described previously (Wu et al., 2010 (link)). Briefly, 10-μm retinal cryosections were fixed with 4% paraformaldehyde for 20 min at room temperature and then incubated with 0.1% Triton X-100 and 3% bovine serum albumin (BSA) for 40 min at room temperature. Sections were then incubated with a rabbit anti-caspase-3 antibody (1:300 dilution, Abcam) overnight before being incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:500, Invitrogen-Molecular Probes, Carlsbad, CA, United States) for 1 h at room temperature. Sections were then counterstained with Hoechst 33258 (1:1000, Invitrogen-Molecular Probes) and visualized and photographed using a confocal microscope (Leica SP8).
7
Cellular Localization of BT1636 in Bacteroides
8
Visualizing Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NETs were stained for immunofluorescence microscopy as described (32 (link)) using methodology modified from (33 (link)). In brief, 5 ×105 neutrophils were added to fibrinogen-coated coverslips, stimulated for 4 h with 40 nM PMA, 0.5 mM MnCl2 or varying concentrations of leukadherin-1 (LA-1; Sigma, UK), and fixed with 4% PFA. Coverslips were blocked and sequentially incubated with an anti-histone H3 antibody (Abcam, UK) and Alexa Fluor® 488-conjugated goat anti-rabbit IgG secondary antibody (Life Technologies, UK). Coverslips were washed, mounted, and sealed using with ProLong™ Gold antifade mountant with DAPI (Invitrogen, UK). Slides were visualized using a Zeiss Axio Imager.A1 inverted fluorescence microscope (Zeiss, Germany) and images analyzed using Image J.
9
Neutrophil Histone H3 Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glass coverslips (Fischer, UK) were sterilized with and coated with 200 μg/ml fibrinogen (Sigma, UK) at 4 °C. 5 × 105 neutrophils were then added to coverslips and stimulated for 4 hours, after which they were fixed for 15 minutes with 4% PFA at room temperature. Coverslips were blocked in a blocking solution (10% goat serum/1% BSA/2 mM EDTA/HBSS/0.1% Tween-2) overnight at 4 °C, then incubated with 1 µg/ml anti-histone H3 antibody (ab1791, Abcam, UK) and then 2 µg/ml Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Life Technologies, UK), which were diluted in blocking solution, for one hour each at room temperature. After incubations, coverslips were washed with HBSS twice, mounted and sealed on microscope slides with ProLong Gold antifade mountant with DAPI (Invitrogen, UK). Slides were subsequently visualized using a Zeiss Axio Imager.A1 inverted fluorescence microscope (Zeiss, Germany) and images analysed using Image J.
10
Immunofluorescence Microscopy of NETs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NETs were stained for immunofluorescence microscopy as described 73 (link) . In brief, 5x10 5 neutrophils were added to coverslips, stimulated, and then fixed with 4% PFA. Coverslips were blocked and sequentially incubated with an anti-histone H3 antibody (Abcam, UK) and Alexa Fluor® 488-conjugated goat anti-rabbit IgG secondary antibody (Life Technologies, UK).
Coverslips were washed, mounted, and sealed using with ProLong™ Gold antifade mountant with DAPI (Invitrogen, UK). Slides were visualized using a Zeiss Axio Imager.A1 inverted fluorescence microscope (Zeiss, Germany) and images analysed using Image J.
Coverslips were washed, mounted, and sealed using with ProLong™ Gold antifade mountant with DAPI (Invitrogen, UK). Slides were visualized using a Zeiss Axio Imager.A1 inverted fluorescence microscope (Zeiss, Germany) and images analysed using Image J.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!