Goat anti human alexa fluor 488
Goat anti-human Alexa Fluor 488 is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize human target proteins in various applications such as immunohistochemistry, flow cytometry, and Western blotting.
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12 protocols using goat anti human alexa fluor 488
Complement Pathway Activation Analysis
Flow Cytometry for Receptor Expression
Visualizing ADC Internalization in Ramos Cells
Immunofluorescence Analysis of Lysosomal Localization
Hypoxia-Induced CAIX Expression Analysis
in 6-well plates to confluency. To induce CAIX expression, cells were
cultured under hypoxic conditions of 1% O2 for 24, 48,
or 72 h in a Whitley H35 Workstation (Don Whitley Scientific). The
binding of radiolabeled antibodies and 50% inhibitory concentration
(IC50) were determined as described by Heskamp et al.31 (link) The percentage of CAIX+ cells was
determined by flow cytometry analysis on a FACSCanto II (BD Bioscience).
Cells were harvested, and fixable viability dye eFluor 780 (eBioscience,
65-0865-14) was added to distinguish dead and live cells. Cells were
stained with Human IgG1 anti-mouse CAIX (MSC3) for 30 min at 4 °C
and with goat anti-human Alexa Fluor 488 (Invitrogen, A-11013) for
15 min at 4 °C. Data were analyzed using FlowJo V10.7 (Tree Star).
SARS-CoV-2 Spike Protein Immunofluorescence Assay
Axl Expression Analysis in Lung Cancer
nonsmall cell lung cancer cell line NCI-H249, obtained from American
Type Culture Collection (Manassas, VA), were grown in RPMI-1640 supplemented
with 10% fetal bovine serum. For FACS analysis, adherently grown A549
cells were detached by applying nonenzymatical citric saline buffer.21 (link) NCI-H249 cells were grown in suspension. To
minimize nonspecific uptake, the procedures were performed on ice.
Aliquots of cells were blocked with 10% normal goat serum, incubated
with anti-Axl primary antibody h173 (kindly provided by Vasgene Therapeutics
Inc., Los Angeles, CA) produced using composite human antibody technology20 (link) and goat antihuman Alexa Fluor 488 (Invitrogen,
Paisley, Scotland) in sequence. Subsequently, cells were measured
by flow cytometry (CyAn analyzer, Beckman Coulter). Cells without
the incubation of primary antibody were used as negative controls.
SARS-CoV-2 Spike Protein Immunofluorescence
Immunohistochemical Analysis of TBI Samples
Quantifying SARS-CoV-2 Spike Protein Expression
S3 cells at 4 × 105 cell/mL were infected with 10
MOI ChAdOx1 nCoV-19 and incubated at 37 °C for 40–48 h.
Cells were harvested and pelleted by centrifugation at 500g for 5 min. Resuspended cells were split into three pools
for the alternative staining and were incubated with either pooled
prime-boost sera derived from outbred CD1 mice immunized intramuscularly
with 108 infectious units of ChAdOx1 nCoV-19,18 (link) prepared at 1:50 in PBS-BSA-0.5% or human mAbs
prepared at 1 μg/mL or recombinant ACE2 expressed with a human
Fc tag prepared at 2 μg/mL (see below). Cells were incubated
for 2 h at rt. Cells were washed 3× with PBS-BSA-0.5% and then
incubated for 1 h at rt with secondary antibodies conjugated with
Alexafluor488 diluted at 1:1000 in PBS-BSA 0.5% (for ChAdOx1 nCov-19
serum staining Goat Anti mouse AlexaFluor 488 (Life Tech A11029),
for ACE2 staining, Goat Anti Human AlexaFluor 488 (Life Tech)). Cells
were washed twice with PBS-BSA-0.5% before resuspending in PBS and
analyzed by flow cytometry using a Fortessa X20 FACS analyzer. Samples
were considered positive for spike expression if they had a fluorescence
intensity above a threshold value determined by the maximum intensity
of the noninfected control cells (
and representative data are shown. Data were analyzed using FlowJo
v9 (TreeStar).
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