At 36 h post-transfection, HEK293 cells were washed with PBS. To detect GluA2 on the cell surface membrane, cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature and incubated with anti-GluA2 N-terminus monoclonal antibody (Millipore, final concentration 5 μg/ml) in PBS containing 3% bovine serum albumin (BSA) for 1 h at room temperature (nonpermeabilizing conditions). To detect the intracellular GluA2, the cells were then fixed in methanol for 15 min at -20°C and incubated with anti-GluA2/3 polyclonal antibody (Enzo Life Sciences, final concentration 5 μg/ml) in PBS containing 3% BSA for 1 h at room temperature (permeabilizing conditions) followed by incubation with AlexaFluor488-conjugated anti-rabbit IgG antibody and AlexaFluor546-conjugated anti-mouse IgG antibody (Molecular Probes, final concentration 5 μg/ml) in PBS containing 3% BSA for 1 h at room temperature. Intracellular and cell surface GluA2 were observed using the FluoView imaging system (Olympus).
Alexa fluor 488 conjugated anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 488-conjugated anti-rabbit IgG antibody is a secondary antibody that binds to rabbit primary antibodies. The Alexa Fluor 488 dye allows for fluorescent detection and visualization of rabbit target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mowiol, by Merck Group (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Alexa fluor 568 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 510 meta microscope, by Zeiss (2 mentions)
Anti glua2 3 polyclonal antibody, by Enzo Life Sciences (1 mentions)
Fluoview imaging system, by Olympus (1 mentions)
Anti cd68 kp1, by Abcam (1 mentions)
Confocal microscope, by Leica (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
T7471, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Dxm1200c, by Nikon (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Bay11 7082, by Merck Group (1 mentions)
Tcs sp5 aobs tandem, by Leica (1 mentions)
27 protocols using alexa fluor 488 conjugated anti rabbit igg antibody
1
GluA2 Surface and Intracellular Localization
2
Immunostaining of Differentiated THP-1 Monocytes
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THP-1 monocytes were seeded at 100 000 cells/well in 24-well plates containing a coverslip and were differentiated as described here above. Undifferentiated monocytes were attached on coverslips by drying a PBS drop containing 100 000 cells. For labeling, cells were fixed for 10 min with paraformaldehyde 4 % in cold PBS, washed three times with 2 % PBS–BSA (bovine serum albumin) and incubated overnight at 4 °C with the primary antibody 1:100 diluted in 2 % PBS-BSA: anti-CD68 (KP1) from Abcam (ab955), anti-CD71 (H300) from Santa Cruz (sc-9099), anti-CD36 (H300) from Santa Cruz (sc-9154), anti-CD14 (1H5D8) from Abcam (ab181470). Cells were washed three times with 2 % PBS–BSA and then incubated for 1h with the secondary antibody. Alexa Fluor-488-conjugated anti-rabbit IgG antibody (Molecular Probes, #A11034) was used at 1/1000 dilution. Cells were then washed three times with PBS, the coverslips were mounted in Mowiol (Sigma) and observed with a confocal microscope (SP5, Leica).
3
Immunofluorescence Labeling of p65 in Cells
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Immunofluorescence labeling was performed as described before21 (link),22 (link). Briefly, cells were fixed 10 min in 4% paraformaldehyde in PBS. Cells were washed with PBS, then permeabilized with 0.1% Triton X100 in PBS during 5 min. Cells were blocked with 2% BSA in PBS 30 min and incubated O/N with primary antibody at 4 °C (CST #8242; p65; 1:400 diluted in PBS BSA 2%). Cells were rinsed 30 min in PBS BSA 2% and incubated with secondary antibody (Alexa Fluor 488-conjugated anti-rabbit IgG antibody; Molecular Probes, #A11034). Cells were then incubated with TOPRO-3 to stain the nucleus. The coverslips were mounted on Mowiol (Sigma) and the pictures taken with confocal microscope (SP5, Leica).
4
Histological Analysis of Tissue Samples
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Tissue samples were fixed in 3.7% formaldehyde, embedded in paraffin, and cut into 5-μm sections. For histological analysis, sections were stained with hematoxylin and eosin (H&E) or treated with rabbit anti-mouse TM antibody purified by affinity chromatography in our laboratory. Alexa Fluor 488-conjugated anti-rabbit IgG antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody for immunofluorescence staining.
5
Immunofluorescence Assay for NFκB in HUVEC
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HUVEC cells (1 × 104 cells) were cultured on poly-lysine treated cover slips in EGM2 bullet kit (Lonza) for 16 hours and transfected with the miRNA. After 24 hours, cells were treated with LPS for 6 hours. The cells were then fixed in 4% paraformaldehyde for 5 min at 4 °C, permeabilized with 20% v/v Methanol for 5 min at 4 °C, and blocked with 3% normal goat serum (NGS) in 1X PBS containing 0.1% Triton-X-100 for 30 min. The cells were incubated overnight at 4 °C with rabbit anti-NFκB (ab131539, Abcam) in PBS containing 3% NGS and 0.2% Triton-X-100, washed thrice with PBS and blocked again with 10% NGS and washed thrice before staining with Alexafluor 488-conjugated anti-rabbit IgG antibody (A21206, Molecular probes), Texas Red®-X Phalloidin (T7471, Molecular probes) and DAPI for 30 min. The cells were washed twice with PBS and images were acquired using the Olympus FV1000 confocal microscope.
6
Visualizing NF-κB Activation in HEK293T Cells
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HEK293T cells in 8-well glass chamber plate (Nalge Nunc International, Rochester, NY) were transfected with phTLR5 using Fugene 6 (Roche). The cell culture was replaced with fresh DMEM containing PMA for 6 hours. After fixation for 15 minutes with 3.7% paraformaldehyde, the cells were rendered permeable by incubation in PBS with 0.2% Triton X-100 for 10 minutes. NF-κB p65 protein was detected by immunostaining using a specific antibody (Santa Cruz Biotechnology, Delaware Avenue, Santa Cruz, CA) and Alexa Fluor-488-conjugated anti-rabbit-IgG antibody (Molecular Probes, Invitrogen, Eugene, OR). Fluorescence images were acquired using a fluorescence microscope (DXM1200C, Nikon).
7
Immunofluorescence Analysis of Autophagy Markers
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HepG2 cells were seeded at 50 000 cells/well and A549 cells at 30 000 cells/well in 24-well plates 24 h before incubation with HFCP, pectin or etoposide for 24 h. After incubation, the cells were fixed and permeabilized for 10 min with a cold solution of 80% methanol and 20% acetone, rinsed three times with PBS-2% BSA and incubated for 2 h with primary antibodies. The primary antibody for LC3 staining was rabbit anti-LC3 (L7543 Sigma) (1/250 dilution), and the primary antibody for LAMP1 staining was mouse anti-LAMP1 (H4A3 received from August & Hildreth, Baltimore [13 (link)]). The cells were washed three times with PBS-2% BSA and then incubated for 1 h with the secondary antibodies. Alexa Fluor-488-conjugated anti-rabbit IgG antibody and Alexa Fluor-568-conjugated anti-mouse IgG antibody (Molecular Probes) were used at 1/1000 dilution. After 1 h of incubation, the cells were rinsed three times with PBS. For nuclear labeling, the cells were incubated for 30 min with TOPRO–3 (Molecular Probes, dil. 1/80) in the presence of 2 mg/ml RNAse and then rinsed three times with PBS. Finally, coverslips were mounted using Mowiol (Sigma, St Louis, USA), and the cells were observed with a fluorescent confocal microscope (Leica SP5).
8
NF-κB Activation Inhibition in RAW264.7 Cells
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RAW264.7 cells were plated into an 8-well glass chamber plate (Thermo Fisher Scientific, United States) at 1 × 105 cells/mL overnight. The cells were pretreated with ZCO (0.01%) or Bay11-7082 (20 μM) for 2 h prior to stimulation with 1 μg/mL of LPS (Sigma Co., MO, United States) for 2 h, and then fixed with 4% paraformaldehyde (Molecular Probes, Inc., Eugene, OR, United States) and permeabilized with 0.1% triton for 15 min. The NF-κB p65 protein was detected by immunostaining using a polyclonal-anti-NF-κB p65 antibody (Invitrogen, Carlsbad, MA, United States) and Alexa Fluor 488-conjugated anti-rabbit IgG antibody (Molecular Probes Invitrogen, MA, United States). Actin was visualized by staining with Alexa Fluor 594 conjugated phalloidin. The cells were mounted by using Prolong gold anti-fade reagent with DAPI (Molecular Probes). Bay 11-7082 (Sigma, MO, United States) was used as the positive control for the NF-κB inhibitors. Fluorescence images were acquired using Laser Scanning Confocal Microscope System (Leica TCS SP5/AOBS/Tandem, Germany) at Korea Basic Science Institute, Gwangju Center).
9
Quantifying Cell Proliferation in Cricket Regenerating Legs
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Proliferating cells in the S phase were detected using the Click-iT EdU Alexa Fluor 488 Imaging Kit (C10337, Thermo Fisher Scientific). EdU was injected into the abdomen of third instar cricket nymphs at 44 hpa, and the RLs were fixed at 48 hpa (4 h after EdU injection) with 2% paraformaldehyde (PFA) in PBS containing 0.05% Tween-20 (PBT) overnight at 4°C. EdU-incorporated cells were detected according to the manufacturer's instructions. Hoechst 33342 (H3570, Thermo Fisher Scientific) was used for nuclear staining. Proliferating cells in the M phase were detected by immunostaining of phospho-histone H3S10. Regenerating legs at 48 hpa were fixed with 2% PFA and cuticles were removed, then regenerating tibiae were washed with PBT and blocked with 1% normal goat serum (NGS) in PBT for 1 h. Blocked samples were incubated with primary antibody [rabbit polyclonal anti-phospho-histone H3 (Ser10) antibody; 06-570, Millipore] at 1:500 in 1% NGS in PBT overnight at 4°C. The samples were washed with PBT and blocked with 1% NGS in PBT. The samples were incubated with secondary antibody (Alexa Fluor 488-conjugated anti-rabbit IgG antibody; A-11008, Molecular Probes) at 1:750 in 1% NGS in PBT for 3 h at 25°C. Finally, the samples were washed with PBT and incubated with a 1:1000 dilution of Hoechst 33342 in PBT for 15 min.
10
Immunofluorescence Visualization of DDX1 and LGR5
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Cells were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X‐100. Cells were incubated with anti‐DDX1 or anti‐LGR5 (ab75732, Abcam) antibody overnight at 4°C. They were stained with AlexaFluor 488‐conjugated anti‐rabbit IgG antibody (R37116, Thermo Fisher Scientific) and analyzed on a TSC SP8 Confocal microscope (Leica Microsystems, Mannheim, Germany).
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