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Goat anti human alexa fluor 488

Manufactured by Thermo Fisher Scientific
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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

1

Complement Pathway Activation Analysis

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Rabbit polyclonal antibody against the C3d domain of human complement component 3 (C3), and Rabbit polyclonal antibody against the C4c domain of human complement component 4 (C4) were purchased from Dako. Mouse monoclonal anti human C5b-9 antibody directed against a neoepitope exposed on complement component 9 when incorporated into the terminal complement complex (TCC) is from BioPorto Diagnostics. Mouse monoclonal against human complement component 1q (C1q) is from Quidel. Rabbit polyclonal anti SA antibody is from Thermoscientific. Alexa fluor 488 goat anti rabbit IgG, Alexa fluor 594 goat anti mouse IgG and Alexa fluor 488 goat anti human from Molecular Probes. U0126, CRID-3, and AG1478 all used at concentration of (10 µM) from Tocris. Human purified C1q, Mannan-binding lectin (MBL), complement Factor B, C3, C6 proteins and sera depleted of C1q, C3, Factor B, C6 were from Quidel. The MBL deficient serum was obtained from an individual homozygous for the D (R52C, rs5030737) variant not able to activate the MBL dependent lectin pathway has been previously described (26 (link)). IdeS was purified as previously described (27 (link)) and was generously provided by Lars Björck and Inga-Maria Frick.
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2

Flow Cytometry for Receptor Expression

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Flow cytometry for receptor surface expression analysis was performed according to the standard procedures. Around one million of cells were harvested and labeled with EV20 or EV20-Sap on ice for 30 minutes. Cells were then washed with 2 ml PBS, pulled down, and stained with an Alexa Fluor 488 Goat anti-Human (Molecular Probes, Life Technologies). For commercial anti-HER-3 C-17 sc staining, cells were fixed and permabilized before antibody incubation.
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3

Visualizing ADC Internalization in Ramos Cells

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Ramos cells were exposed to ADCT-602 or B12-C220-SG3249 (1 hour at 4°C) and then incubated at 37°C where appropriate. Following the permeabilization of cells using Tween 20 (0.1% volume-to-volume ratio in PBS) for 15 minutes, samples were washed with the PBS, and centrifuged at 4,000 rpm (4°C). Removal of the supernatant was followed by the addition of a rabbit mAb LAMP-1 (1:400; Cell Signaling Technology) for 1 hour on ice. A further washing step was followed by adding both Alexa Fluor 488 goat anti-human (1:200; Thermo Fisher Scientific) for detecting the ADC and Alexa Fluor 568 goat anti-rabbit (1:200; Thermo Fisher Scientific). Both secondary antibody incubations were performed on ice for 1 hour. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific), and then the cytospins of cell samples were prepared. The samples mounted in ProLong Gold Antifade (Life Technologies) and cover-slipped.
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4

Immunofluorescence Analysis of Lysosomal Localization

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SN12C and MDA-MB-468 cells were incubated with either ADCT-601 or B12-PL1601 for 1 hour at 4°C, then incubated at 37°C where appropriate. Cells were fixed in 4% paraformaldehyde and permeabilized using 0.1% volume for volume (v/v) Tween-20 in PBS, washed with PBS, and centrifuged at 4,000 rpm (4°C). After removal of the supernatant, a rabbit mAb against lysosome-associated membrane protein 1 (LAMP-1;1:400; Cell Signaling Technology) was added and the cells were kept on ice for 1 hour before being washed. Secondary antibodies were added to detect ADCT-601 (Alexa Fluor 488 Goat Anti-Human; 1:200; Thermo Fisher Scientific) and lysosomes (Alexa Fluor 568 Goat Anti-Rabbit; 1:200; Thermo Fisher Scientific), and the cells were incubated for 1 hour on ice. After a further wash, nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific), cytospins of cell samples were prepared, and samples were visualized by immunofluorescence microscopy (Zeiss LSM 880).
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5

Hypoxia-Induced CAIX Expression Analysis

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Cells were cultured
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).
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6

SARS-CoV-2 Spike Protein Immunofluorescence Assay

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For the IFA, BHK cells transfected with pCAG-SARS-CoV-2 S-full were fixed with acetone-methanol (1:1) at 4 °C for 20 min. Fixed cells were reacted with the test serum samples, which were diluted at 1:100 with PBS. After an incubation for 1 h, the cells were rinsed with PBS and incubated with goat anti-human Alexa Fluor 488 (Invitrogen). After washing with PBS, staining was observed under a fluorescence microscope.
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7

Axl Expression Analysis in Lung Cancer

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Human small cell lung cancer A549 cell line and
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.
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8

SARS-CoV-2 Spike Protein Immunofluorescence

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Indirect immunofluorescence was performed as previously reported (32 (link)). In brief, 36 h after transfection, coverslips were washed three times with PBS, fixed with 4% (w/v) paraformaldehyde in PBS for 10 min at room temperature, rinsed three times with PBS, and permeabilized with 0.1% (v/v) Triton-X100 (Sigma) in PBS for 15 min at room temperature. Next, 1 ml 1% BSA/PBS was added to the coverslips for 30 min at room temperature to block unspecific binding of antibodies. Primary antibodies (rabbit anti-SARS-CoV-2 spike protein monoclonal antibody, 40150-R007, Sino Biological, 1/100 dilution; human anti-SARS-CoV-2 4A8 monoclonal antibody, CPC514A, Cell Sciences, 1/100 dilution in PBS with 1% BSA) were added and incubated at 4°C overnight. Coverslips were washed with PBS three times, and secondary antibodies (Goat anti-Rabbit Alexa Fluor Plus 555 Invitrogen, and Goat anti-Human Alexa Fluor 488, Invitrogen, 1/400 dilution in PBS with 1% BSA) were added and incubated at room temperature for 1 h. Coverslips were washed with PBS three times and counterstained with DAPI (Invitrogen). Immunofluorescence images were acquired with a Leica SP8 X Confocal Microscope (Lecia) using LAS X software.
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9

Immunohistochemical Analysis of TBI Samples

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Animals were anesthetized with a lethal dose of beuthanasia-D solution and transcardially perfused with 4% paraformaldehyde. Whole brains were removed, processed, embedded in paraffin, and cut into 4–6 μm sections. After de-parafinization, slides were incubated for 10 min at 95°C in Trilogy solution (Cell Marque), blocked with 3% hydrogen peroxide, followed by 2% normal goat serum. The sections were incubated with TBI or normal human serum overnight at 4°C, then incubated with goat anti-human HRP (Abcam) diluted in 2% goat serum. Staining was visualized with 3,3′-diaminobenzidine (DAB; Dako). The sections were counterstained with hematoxylin (Dako). Sections were finally washed with PBS, mounted, air-dried and cover slipped with Aquamount (Dako). The slides were scanned and examined using the Aperio ScanScope GL system at 20 x and ScanScope software. For immunofluorescence experiments, sections were incubated with anti-GFAP Alexa Fluor 555 conjugate (Cell Signaling) and either normal or TBI human serum overnight at 4°C, followed by goat anti-human Alexa Fluor 488 (Life Technologies) diluted in 2% goat serum. The sections were washed with PBS, mounted, air-dried and cover slipped with FluoroGel (GenTex). Staining was imaged with a Zeiss fluorescence microscope.
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10

Quantifying SARS-CoV-2 Spike Protein Expression

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Hela
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 (Figure 1D). Experiments were performed twice in duplicate,
and representative data are shown. Data were analyzed using FlowJo
v9 (TreeStar).
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