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Secondary antibody conjugated to alexa fluor 488

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The secondary antibody conjugated to Alexa Fluor 488 is a fluorescent labeling reagent. It is designed to bind to and detect primary antibodies, enabling visualization and quantification of target molecules in various applications, such as immunofluorescence, flow cytometry, and Western blotting.

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Lab products found in correlation

Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions) M csf, by Thermo Fisher Scientific (2 mentions) Fibronectin coated cover slips, by R&D Systems (2 mentions) Paraformaldehyde 4, by Thermo Fisher Scientific (2 mentions) Primary anti vinculin antibody, by Merck Group (2 mentions) Phalloidin conjugated to alexa fluor 633, by Thermo Fisher Scientific (2 mentions) Triton, by Merck Group (2 mentions) Magnetic bead, by Miltenyi Biotec (2 mentions) Cell dissociation solution, by Merck Group (1 mentions) Flojo flow cytometry analysis software, by Tree Star (1 mentions) Facscan caliber flow cytometer, by BD (1 mentions) Cryostat microtome, by Leica (1 mentions) Structured illumination, by Zeiss (1 mentions) Triton x 100, by Thermo Fisher Scientific (1 mentions) F actin, by Abcam (1 mentions) Mounting medium, by Vector Laboratories (1 mentions) Goat serum, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Dm5500b upright microscope, by Leica (1 mentions) Image pro plus software, by Media Cybernetics (1 mentions)

Close spelling variants of this product

Donkey antimouse secondary antibody conjugated to Alexa Fluor 488 Goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 Donkey anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 Goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 Goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 Alexa Fluor 488-conjugated secondary antibody Alexa Fluor 488 secondary antibody Alexa Fluor 488-conjugated antibody Alexa 488-conjugated secondary antibody Alexa Fluor-conjugated secondary antibodies

8 protocols using secondary antibody conjugated to alexa fluor 488

1

Visualizing Listeria Monocytogenes Invasion

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Caco‐2 cells were infected with LM as described in the invasion analysis assay at an MOI of 100:1 with or without baicalein addition. At 1 h post‐infection (pi), infected Caco‐2 cells were washed three times with PBS, fixed with 4% paraformaldehyde for 20 minutes and incubated with blocking solution (5% BSA diluted in PBS) for 1 h. Extracellular bacteria were stained with LM‐specific antibody (Abcam) and secondary antibody conjugated to Alexa Fluor 488 (Invitrogen). Following permeabilization with 0.3% Triton X‐100 for 3 minutes, both intracellular and extracellular bacteria were stained with the same primary antibody and a secondary antibody conjugated to Alexa Fluor 594 (Molecular Probes). The images were captured using a confocal laser scanning microscope.
Lu G., Xu L., Zhang T., Deng X, & Wang J. (2018). A potential bio‐control agent from baical skullcap root against listeriosis via the inhibition of sortase A and listeriolysin O. Journal of Cellular and Molecular Medicine, 23(3), 2042-2051.
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2

Flow Cytometry Sample Preparation

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Monolayers of cells were washed once with PBS containing 0.04% EDTA and incubated with cell dissociation solution (Sigma) to remove cells from plates. Cells were washed with PBS and fixed in 4% paraformaldehyde for 10 minutes on ice. Cells were then pelleted and resuspended in blocking buffer (Tris-buffered saline (TBS) with 1% BSA) for 20 min on ice. Cells were pelleted and incubated with primary antibody or IGG control prepared in TBS with 1% BSA for 30 min on ice. Following incubation, cells were washed twice with TBS/1% BSA and then incubated with secondary antibody conjugated to Alexafluor 488 (Invitrogen) for 30 min on ice. The stained cells were washed twice with TBS containing 1% BSA, resuspended in TBS containing 1% BSA, and analyzed by FACScan caliber flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Data was analyzed using FloJo flow cytometry analysis software (Tree Star, Inc.; Ashland, OR).
DiMaio T.A., Wentz B.L, & Lagunoff M. (2015). Isolation and Characterization of Circulating Lymphatic Endothelial Colony Forming Cells. Experimental cell research, 340(1), 159-169.
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3

Visualizing Astrocyte-Derived Extracellular Vesicles

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EVs released from astrocytes of GFAP-EGFP mice were visualized by fluorescence imaging of EGFP+ puncta in liver, spleen, and lung tissue sections. Tissues were postfixed in 4% PFA and cryoprotected in 30% sucrose, and 20-µm sections were cut using a cryostat microtome (Leica). Nonspecific binding was blocked with 5% normal goat serum plus 5% normal horse serum in tris-buffered saline (TBS) containing 0.1% Triton X-100 (Fisher Scientific). EGFP was enhanced by incubating sections with anti-GFP rabbit sera (1:500; Invitrogen) at 4°C overnight, followed by incubation with a secondary antibody conjugated to Alexa Fluor 488 (1:1000; Invitrogen). Cells were visualized with F-actin (1:100; Abcam), and nuclei were stained with DAPI as previously described (60 (link)). Imaging was performed with 40× and 100× objectives using optical sectioning by structured illumination (Carl Zeiss Inc.).
Dickens A.M., Tovar-y-Romo L.B., Yoo S.W., Trout A.L., Bae M., Kanmogne M., Megra B., Williams D.W., Witwer K.W., Gacias M., Tabatadze N., Cole R.N., Casaccia P., Berman J.W., Anthony D.C, & Haughey N.J. (2017). Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions. Science signaling, 10(473), eaai7696.
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4

Immunofluorescence and Western Blot Analysis of mGlu1 Receptors

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For immunofluorescence analysis, 30-lm retinal sections were incubated with a mouse anti-mGlu1 primary antibody (1:200, BD Bioscience, Milan, Italy). Serial sections were then incubated with a secondary antibody conjugated to Alexa Fluor 488 (1:200, Invitrogen, Carlsbad, CA, USA). Slides were coverslipped with mounting medium (Vector). Tissue sections were scanned using a LSM 5 Pascal confocal laser scanning microscope with a Zeiss ECPLAN-NEOFLUAR 40Â/1.30 M27 oil immersion objective (Carl Zeiss Microimaging Inc.). We used a 488-nm argon laser to excite Alexa Fluor 488.
Western blot analysis of mGlu1a receptors in crv4 and wild-type mice Immunoblot analysis of mGlu1a receptors was carried in the cerebellum of crv4 mice and their wild-type littermates as reported previously (Romano et al., 2016) using a mouse monoclonal anti-mGlu1a antibody (1:700, BD Biosciences).
Liberatore F., Bucci D., Mascio G., Madonna M., Di Pietro P., Beneventano M., Puliti A.M., Battaglia G., Bruno V., Nicoletti F, & Romano M.R. (2017). Permissive role for mGlu1 metabotropic glutamate receptors in excitotoxic retinal degeneration. Neuroscience, 363.
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5

Isolation and Characterization of Monocyte-Derived Macrophages

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Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors and patients were thawed and CD14+ cells selected using magnetic beads (Miltenyi). 2 x 105 cells/ well in a 24 well plate were seeded on 10ug/ml fibronectin-coated cover slips (R&D systems) in 500ul 20ng/ml macrophage colony stimulating factor (MCSF, Gibco) for 6 days to obtain monocyte-derived macrophages (MDMs). Cells were fixed with paraformaldehyde 4% (Thermo Fisher Scientific) for 10 minutes on ice followed by 8% for 20 minutes at room temperature, permeabilised with 0.1% triton (Sigma) for 5 minutes at room temperature and non-specific binding reduced by blocking with 5% BSA/PBS for 1 hour at room temperature. Cells were incubated with primary anti-vinculin antibody (Sigma 1:200) for 1 hour at room temperature, washed twice with PBS and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:500 Life Technologies) and phalloidin-conjugated to Alexa Fluor 633 (1:200 Thermo Fisher Scientific) for one hour at room temperature. Cells were washed twice with PBS and cover slips mounted onto slides using mounting solution with DAPI for nuclear staining (ProLong Diamond Antifade Mountant with DAPI, Life Technologies) overnight. Slides were imaged using Zeiss 710 confocal microscope at 63x magnification and podosome analysis was carried out on at least 100 cells per sample from 10 fields of view.
Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.
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6

Isolation and Characterization of Monocyte-Derived Macrophages

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Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors and patients were thawed and CD14+ cells selected using magnetic beads (Miltenyi). 2 x 105 cells/ well in a 24 well plate were seeded on 10ug/ml fibronectin-coated cover slips (R&D systems) in 500ul 20ng/ml macrophage colony stimulating factor (MCSF, Gibco) for 6 days to obtain monocyte-derived macrophages (MDMs). Cells were fixed with paraformaldehyde 4% (Thermo Fisher Scientific) for 10 minutes on ice followed by 8% for 20 minutes at room temperature, permeabilised with 0.1% triton (Sigma) for 5 minutes at room temperature and non-specific binding reduced by blocking with 5% BSA/PBS for 1 hour at room temperature. Cells were incubated with primary anti-vinculin antibody (Sigma 1:200) for 1 hour at room temperature, washed twice with PBS and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:500 Life Technologies) and phalloidin-conjugated to Alexa Fluor 633 (1:200 Thermo Fisher Scientific) for one hour at room temperature. Cells were washed twice with PBS and cover slips mounted onto slides using mounting solution with DAPI for nuclear staining (ProLong Diamond Antifade Mountant with DAPI, Life Technologies) overnight. Slides were imaged using Zeiss 710 confocal microscope at 63x magnification and podosome analysis was carried out on at least 100 cells per sample from 10 fields of view.
Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.
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7

Immunofluorescent Staining of E-Cadherin

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For staining of E-cadherin protein, the cells were fixed in 4% paraformaldehyde/PBS for 20 min and permeabilized with 0.1% Triton X-100/PBS for 3 min at room temperature. Subsequently, cells were blocked in 2% goat serum plus 1% BSA (both from Thermo Fisher Scientific, Inc.) in PBS for 30 min at room temperature, incubated with primary antibody at 4°C overnight, and then incubated with secondary antibody conjugated to Alexa Fluor 488 (cat. no. A32731; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. The nuclei were counterstained with DAPI. The primary antibody used was anti-E-cadherin (1:200; cat. no. ab40772; Abcam). Images were acquired using a light Leica DM5500B upright microscope (Leica Microsystems GmbH) with a Retiga SRV Cooled CCD camera (Teledyne Photometrics) and ImagePro Plus software (version 6.0; Media Cybernetics, Inc.).
Liang M., Zhu B., Wang M, & Jin J. (2022). Knockdown of long non-coding RNA DDX11-AS1 inhibits the proliferation, migration and paclitaxel resistance of breast cancer cells by upregulating microRNA-497 expression. Molecular Medicine Reports, 25(4), 123.
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8

Quantifying Activated STAT3 in Hypothalamus

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The hypothalamus sections were fixed in 10% formalin (10 min), washed in PBS (5 × 8 min), and then incubated with 1% H2O2 and 0.3% NaOH (20 min), 0.3% glycine (10 min), and 0.03% sodium dodecyl sulfate (SDS; 10 min). After rinsing in PBS, the sections were blocked with 10% normal goat serum (in 0.3% Triton X-100; 60 min) and incubated overnight at 4 °C with the primary p-STAT3 (Y705) antibody (Cell Signaling Technology, Boston, MA, USA), diluted 1:300 in a block solution. After thorough rinsing in PBS (3 × 8 min), the sections were incubated with secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:600 in block solution overnight at 4 °C. Then, the preparations were washed in PBS (3 × 8 min) and mounted in Vectashield mounting medium with DAPI (Vector Labs, Burlingame, CA, USA) under a cover glass. The sections were viewed using an inverted microscope Eclipse Ni (Nikon, Tokyo, Japan), and pictures were taken. The percentage of p-STAT3-positive nuclei to the total number of nuclei was calculated using ImageJ software (NIH).
Mitkin N.A., Pavshintcev V.V., Sukhanova I.A., Doronin I.I., Babkin G.A., Sadagurski M, & Malyshev A.V. (2022). The Novel Peptide Chm-273s Has Therapeutic Potential for Metabolic Disorders: Evidence from In Vitro Studies and High-Sucrose Diet and High-Fat Diet Rodent Models. Pharmaceutics, 14(10), 2088.
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