Approximately 106 dissociated sgCtrl and UGCG-KO cells (~106) were washed twice with fluorescence-activated cell sorting (FACS) buffer (1x PBS, 0.2% sodium azide, and 1% FBS) and transferred to the wells of a 96 well U-bottom plate, then incubated with 1:200 dilution of Alexa-488 conjugated Cholera toxin subunit B (diluted in FACS buffer) for 1 hr on ice in the dark. As an isotype control, sgCtrl cells were separately incubated with 1:200 dilution of Alexa 488 anti-rabbit antibody (Thermo Scientific) for 1 hr on ice. Following three washes with FACS buffer, cells were passed through a 35 μm cell strainer attached to a 5 ml polystyrene tube to prevent clumping (#352235, BD Biosciences). All samples were kept on ice until subjected to flow cytometry using a FACSCalibur cytometer (BD Biosciences). Data were analyzed with FlowJo 9.9.6 software (BD Biosciences).
Alexa488 anti rabbit antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa488 anti-rabbit antibody is a fluorescent-labeled secondary antibody used for immunodetection. It is designed to bind to rabbit primary antibodies, allowing for the visualization of target proteins in various applications such as western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Microscope slide, by Thermo Fisher Scientific (3 mentions)
24 well plate, by Greiner (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Lsm 700 confocal microscope, by Zeiss (3 mentions)
Facscalibur cytometer, by BD (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Accuri c6, by BD (2 mentions)
Vectashield with dapi, by Vector Laboratories (2 mentions)
Autoclaved 12mm circle micro cover glass, by Avantor (2 mentions)
Fmoc l histidine amino acids, by Merck Group (2 mentions)
Setd3, by Abcam (2 mentions)
Pan actin, by Cytoskeleton (2 mentions)
β tubulin, by Merck Group (2 mentions)
H3k4me2, by Cell Signaling Technology (2 mentions)
H3k36me2, by Cell Signaling Technology (2 mentions)
Eclipse ti confocal microscope, by Nikon (1 mentions)
Bx60 fluorescence microscope, by Olympus (1 mentions)
Las af imaging software, by Leica (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
16 protocols using alexa488 anti rabbit antibody
1
Quantifying Cholera Toxin Binding in UGCG-Deficient Cells
2
Quantifying Cholera Toxin Binding in UGCG-Deficient Cells
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Approximately 106 dissociated sgCtrl and UGCG-KO cells (~106) were washed twice with fluorescence-activated cell sorting (FACS) buffer (1x PBS, 0.2% sodium azide, and 1% FBS) and transferred to the wells of a 96 well U-bottom plate, then incubated with 1:200 dilution of Alexa-488 conjugated Cholera toxin subunit B (diluted in FACS buffer) for 1 hr on ice in the dark. As an isotype control, sgCtrl cells were separately incubated with 1:200 dilution of Alexa 488 anti-rabbit antibody (Thermo Scientific) for 1 hr on ice. Following three washes with FACS buffer, cells were passed through a 35 μm cell strainer attached to a 5 ml polystyrene tube to prevent clumping (#352235, BD Biosciences). All samples were kept on ice until subjected to flow cytometry using a FACSCalibur cytometer (BD Biosciences). Data were analyzed with FlowJo 9.9.6 software (BD Biosciences).
3
Nanoparticle Uptake and Gli2 Expression
4
Immunolabeling and Confocal Imaging of Adult Brains
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Adult brains were dissected in phosphate buffered saline (PBS), fixed for 15 min in 4% paraformaldehyde, washed in PBT (0.5% Triton X-100 in PBS), stained in primary antibodies at 4°C for ca. 48 h, washed in PBT, stained in fluorescent secondary antibodies at 4°C for overnight, then washed in PBT and mounted using Vectashield (Vector Laboratories, Inc.). A Leica TCS SP5 confocal microscope was used to capture sequential images of the brains at 20× magnification with LAS AF imaging software from Leica (version 2.6.0). Anti-TH mouse monoclonal antibody used in these experiments was obtained from Immunostar (Hudson, WI, USA), and was used at a concentration of 1:500. Anti-GFP rabbit polyclonal antibody was procured from Santa Cruz Biotechnology, Inc., and was also used at a concentration of 1:500. Alexa633 anti-mouse antibody and alexa488 anti-rabbit antibody (Invitrogen) were both used as secondary antibodies at 1:200. All antibodies were diluted in PBT+10% normal goat serum (NGS Jackson ImmunoResearch Laboratories).
5
Quantifying Cofilin Phosphorylation in Breast Cancer Cells
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MCF7 human breast cancer cells were plated at 5×103 cells/well in black polystyrene glass-bottomed 96 well plates and treated the next day with 0.2 μg/mL doxorubicin plus indicated LIMK inhibitor concentrations for 18 hours. Cells were washed, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (v/v) and blocked for 1 hour with 1% BSA (w/v). Fixed cells were stained with rabbit antibody against phospho-Ser3 cofilin, then Alexa 488 anti-rabbit antibody (Invitrogen), Texas red Phalloidin (Molecular Probes) and DAPI (Sigma). After washing, cells were imaged on a High Content Imaging Operetta system (PerkinElmer) and phospho-Ser3 cofilin fluorescence intensity for each cell quantified using Harmony® High Content Imaging and Analysis Software (PerkinElmer), and plotted as percent change from DMSO alone-treated control for eight independent replicate determinations. EC50 values were calculated from dose-response curves using Prism 5 (GraphPad).
6
Visualizing Quail and Chick Eye Development
7
Immunofluorescent Staining of Alpha-Synuclein Filaments
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Cells were fixed with 4% PFA in PBS for 15 min, then washed 3 × 5 minutes in PBS. Next, the cells were blocked with 2% BSA + 0.1% Triton-X in PBS for 30 min. The cells were subsequently incubated with anti-alpha synuclein filament antibody (1:1000, Abcam, Cambridge, MA, USA) which is a conformation specific antibody and specifically detects alpha-synuclein filaments or an anti-GABAA receptor alpha 1 (Abcam, Cambridge, MA, USA) primary antibody at 1:250 in 0.1% BSA in PBS. Cells were washed 3 × 5 minutes in PBS, after which they were labelled with Alexa 488 anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA) at 1:1000 for 30 min at room temperature in 0.1% BSA in PBS. Cells were washed 3 × 5 minutes in PBS then imaged using the EVOS FL Imaging System (Invitrogen, Carlsbad, CA, USA).
8
Antibody Staining and Western Blotting Protocol
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Primary mouse antibodies used for western blotting and immunostaining were rat anti-K8 and rat anti-K19 (Troma I respectively Troma III, Hybridoma bank, Iowa, USA), mouse anti-K7 (RCK 105, Abcam, Cambridge, UK), mouse anti-K20 (IT-Ks 20.10, Progen, Frankfurt, De), rat anti-K18 (Troma II, Hybridoma bank, Iowa, USA) [24 (link)], mouse anti-tubulin (Sigma, Munich, Germany), rabbit anti-caspase-7 and anti-cleaved caspase-7 (Cell Signaling, Danvers, MA, USA), rabbit anti-MPO (Thermo Scientific, Waltham, MA, USA) and rat anti-Hsc70 (Stressgen, Victoria, Canada). The secondary antibodies used for staining were Alexa 488 or Alexa 546 anti-mouse, Alexa 488 anti-rat and Alexa 488 anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA). The secondary antibodies used for western blotting were: anti-mouse HRP (GE Healthcare, Little Chalfont, UK), anti-rabbit HRP (Cell Signaling, Danvers, MA, USA) and anti-rat HRP (GE Healthcare, Little Chalfont, UK) antibodies. Nuclei were stained with Draq5 (Cell Signaling, Danvers, MA, USA) or Dapi (Invitrogen Carlsbad, CA, USA). Antibodies used for FACS analysis were anti-CD4-FITC and anti-CD49d-PE or anti-L-selectin-PE (Immunotools, Friesoythe, Germany).
9
Quantifying ADC Internalization in Cancer Cells
10
Quantifying ADC Internalization in Cancer Cells
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Cancer cells were plated in 48-well plates and incubated overnight in a
37 °C incubator with 5% CO2, followed by treatment with 10 nM
Alexa 488-labeled ADC in medium (pH 7.0) for 0.5, 4, and 20 hours. Treated cells
were cooled on ice and surface fluorescence of Alexa 488-labeled ADC quenched
with 5 μg/ml rabbit anti-Alexa 488 antibody (Thermo Fisher Scientific)
for 30 minutes at 4 °C. The quenching efficiency was determined using
separate cell samples which had been incubated with Alexa 488-labeled ADC on ice
(to prevent internalization) followed by treatment with anti-Alexa 488 antibody
or vehicle control. Samples were washed, harvested by trypsinization,
resuspended in PBS, analyzed using a BD Accuri C6 flow cytometer (BD
Biosciences, San Jose, CA, USA) and data processed with FlowJo (FlowJo, Ashland,
OR, USA).
37 °C incubator with 5% CO2, followed by treatment with 10 nM
Alexa 488-labeled ADC in medium (pH 7.0) for 0.5, 4, and 20 hours. Treated cells
were cooled on ice and surface fluorescence of Alexa 488-labeled ADC quenched
with 5 μg/ml rabbit anti-Alexa 488 antibody (Thermo Fisher Scientific)
for 30 minutes at 4 °C. The quenching efficiency was determined using
separate cell samples which had been incubated with Alexa 488-labeled ADC on ice
(to prevent internalization) followed by treatment with anti-Alexa 488 antibody
or vehicle control. Samples were washed, harvested by trypsinization,
resuspended in PBS, analyzed using a BD Accuri C6 flow cytometer (BD
Biosciences, San Jose, CA, USA) and data processed with FlowJo (FlowJo, Ashland,
OR, USA).
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