The cells subjected to various treatments (in 24-well plates) were rinsed thrice with PBS and fixed using 4% cold PBS-buffered paraformaldehyde, followed by permeabilization with 0.2% Triton X-100. The cells were then incubated and blocked with the blocking solution (5% BSA/PBS), followed by incubation overnight at 4°C with the primary antibody—rabbit anti-NF-κB (1:1000, Cell Signaling Technology). Thereafter, the cells were incubated with Alexa Fluor® 488-Conjugated Goat anti-Rabbit IgG (H+L) (1:100,ZSGB-BIO, China) for 1 h in the cassette after washing them with PBS; they were then dyed with 10 μg/mL ready-to-use DAPI solution (Solarbio) for 3 min to stain the nuclei. Representative areas were photographed using a laser scanning confocal microscope (Zeiss, Germany). The experiment was repeated thrice.
Alexa fluor 488 conjugated goat anti rabbit igg h l
Manufactured by ZSGB-BIO
Sourced in China
Alexa Fluor® 488-Conjugated Goat anti-Rabbit IgG (H+L) is a secondary antibody conjugated with the Alexa Fluor® 488 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Confocal laser scanning microscope, by Zeiss (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rabbit anti nf κb, by Cell Signaling Technology (1 mentions)
Dapi solution, by Solarbio (1 mentions)
Propidium iodide rnase staining buffer, by BD (1 mentions)
P iκb, by Cell Signaling Technology (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Protein disulfide isomerase, by Cell Signaling Technology (1 mentions)
Sqstm p62, by Cell Signaling Technology (1 mentions)
Bay11 7082, by Selleck Chemicals (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
5 protocols using alexa fluor 488 conjugated goat anti rabbit igg h l
1
Immunofluorescent Analysis of NF-κB
2
Autophagy and Inflammation Modulation in Cells
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Lipofectamine™ 3000 transfection reagent, TRIzol reagent, LysoTracker Red and the high capacity cDNA reverse transcription kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rapa and 3-MA were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). CQ and BAY-11-7082 were obtained from Selleck (Houston, Texas, USA). Mouse anti-human monoclonal antibodies against PreS2/S, β-actin, and histone 3 were purchased from Abcam (C ambridge, UK). Anti-human monoclonal antibodies against LC3, Beclin-1, SQSTM/P62, NF-κB, p-NF-κB/p65, IκB, p-IκB, Vimentin, E-cadherin and Protein Disulfide Isomerase (PDI) were purchased from Cell Signaling Technology (Boston, MA, USA). The Immobilon Western Chemiluminescent HRP substrate was purchased from EMD Millipore (Billerica, MA, USA). The cell counting kit-8 was purchased from Beyotime (Shanghai, China). Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L), Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L), DyLight 405-labeled goat anti-mouse IgG (H + L) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) were purchased from ZSGB-BIO (Beijing, China). Propidium Iodide (PI)/RNase staining buffer was purchased from BD Biosciences (Franklin, NJ, USA).
3
Immunofluorescence Staining for TLR4 and NF-κB
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Cells deposited
on glass-bottom dishes were rinsed with 1× PBS twice, fixed in
4% (w/v) paraformaldehyde for 20 min, permeabilized with 0.1% (w/v)
Triton X in PBS, washed, and blocked with 1% (w/v) bovine serum albumin
(Sigma-Aldrich) in PBS for 60 min. TLR4 antibody (1:100) (Abcam; ab22048)
or NF-κB p65 antibody (1:400) (Cell Signaling, D14E12) (UniProtKB
Q04206) served as primary reagents. Alexa Fluor 488-conjugated goat
antirabbit IgG (H + L) (ZSGB-BIO, ZF-0511) and Alexa Flour 488-conjugated
goat antimouse IgG (H + L) (ZSGB-BIO, ZF-0512) served as secondary
reagents. Cells were incubated with primary antibodies in 1% bovine
serum albumin in a moist chamber at 4 °C overnight and the secondary
antibodies (1:400) for 1 h at room temperature (RT) in dark. After
DAPI staining, cells were observed with a confocal laser scanning
microscope (Leica, Wetzlar, Germany).
Paraffin sections of recurrent
tumors were rinsed with 1× PBS three times, permeabilized, blocked,
and incubated with antibodies according to the above procedure. TLR4
antibody (1:100) (Abcam; ab22048) served as primary reagents, and
Cy3-conjugated goat antimouse IgG (H + L) (Servicebio; GB21301) served
as secondary reagents. After DAPI staining, TLR4 expression of tumor
sections was observed with a confocal laser scanning microscope (LSM
800, Zeiss, Germany).
on glass-bottom dishes were rinsed with 1× PBS twice, fixed in
4% (w/v) paraformaldehyde for 20 min, permeabilized with 0.1% (w/v)
Triton X in PBS, washed, and blocked with 1% (w/v) bovine serum albumin
(Sigma-Aldrich) in PBS for 60 min. TLR4 antibody (1:100) (Abcam; ab22048)
or NF-κB p65 antibody (1:400) (Cell Signaling, D14E12) (UniProtKB
Q04206) served as primary reagents. Alexa Fluor 488-conjugated goat
antirabbit IgG (H + L) (ZSGB-BIO, ZF-0511) and Alexa Flour 488-conjugated
goat antimouse IgG (H + L) (ZSGB-BIO, ZF-0512) served as secondary
reagents. Cells were incubated with primary antibodies in 1% bovine
serum albumin in a moist chamber at 4 °C overnight and the secondary
antibodies (1:400) for 1 h at room temperature (RT) in dark. After
DAPI staining, cells were observed with a confocal laser scanning
microscope (Leica, Wetzlar, Germany).
Paraffin sections of recurrent
tumors were rinsed with 1× PBS three times, permeabilized, blocked,
and incubated with antibodies according to the above procedure. TLR4
antibody (1:100) (Abcam; ab22048) served as primary reagents, and
Cy3-conjugated goat antimouse IgG (H + L) (Servicebio; GB21301) served
as secondary reagents. After DAPI staining, TLR4 expression of tumor
sections was observed with a confocal laser scanning microscope (LSM
800, Zeiss, Germany).
4
WSSV Infection Assay in Hpt Cells
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The Hpt cell cultures were seeded into cover slides and cultured in 200 μL of L15 medium. After treatment with drugs, Hpt cells were infected with WSSV (MOI = 100) and incubated for 24 h at 26 °C. Culture medium was removed from cover slides and washed with CPBS to clear residual cell debris. Cells were fixed with 4% paraformaldehyde for 10 min at 4 °C and permeated with TritonX-100 for 30 min at room temperature. Subsequently, the cells were blocked for 1 h at room temperature with 5% goat serum dissolved in CPBS. After washing with CPBS, the cells were incubated with anti-SIRT1 polyclonal antibody (ABclonal, Wuhan, China) and anti-VP28 monoclonal antibody at a dilution of 1:300 with CPBS for 2 h at room temperature. The cells were then rinsed with CPBS three times and incubated with Alexa Fluor488 conjugated goat anti rabbit IgG (H + L) and Alexa Fluor 594 conjugated goat anti mouse IgG (H + L) (ZS-Bio, Beijing, China) at a dilution of 1:500 with CPBS for 2 h at room temperature. After a final wash, the cell nuclei were stained with 4′, 6′-diamidino-2′-phenylindole (DAPI) (Beyotime Biotechnology, Shanghai, China) for 10 min at room temperature and analyzed under a confocal laser scanning microscope (Carl Zeiss, Jena, Germany).
5
Immunocytochemistry Staining of PTEN in FLS
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The FLS were plated at a density of 1.0-2.0×10 5 cells/ml in 6-well plates for 24 h. After FLS treatment with TNF-α, immunocytochemistry staining was performed with rabbit anti-PTEN. And the Alexa Fluor 488-Conjugated Goat anti-rabbit IgG (H+L) (ZSGB-BIO, Beijing, China) and 4', 6-diamidino-2-phenylindole (DAPI; Beyotime, China) were incubated in dark. And then the cells were photographed under an Olympus BX-51 microscope (Olympus, Tokyo, Japan).
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