Alexa fluor 488 dye

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada, Switzerland

Alexa Fluor 488 dye is a fluorescent label commonly used in biological research. It is an organic fluorophore with excitation and emission wavelengths of 494 nm and 519 nm, respectively, making it suitable for detection in the green fluorescence channel. Alexa Fluor 488 dye can be conjugated to various biomolecules, such as antibodies, proteins, and nucleic acids, to enable fluorescent labeling and visualization in a variety of applications.

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Lab products found in correlation

156 protocols using alexa fluor 488 dye

Lipid Insertion and Antibody Accessibility in M21 Cells

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Example 4

As an initial assessment of antigen-lipid insertion into cell membranes, a hapten-lipid conjugate, DNP-lipid (100, a dipalmitoleoyl phosphatidylethanolamine-DNP conjugate) and anti-DNP-antibody were used to determine if (1) lipids would insert into model M21 cell line and (2) if the antigen headgroups of the antigen-lipid conjugates would remain accessible to antibodies. M21 cells treated with the DNP-lipid followed by staining with anti-DNP were analyzed by flow cytometry (FIG. 4). The positive shift observed indicated that the lipids had successfully inserted into the cell membrane and that antigen head groups were accessible to antibody.

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To estimate lipid insertion levels into model cells, three fluorescent lipid derivatives were synthesized: 101 an Alexa-Fluor® 488 dye conjugated to cholesterol, 102 Alexa-Fluor® 488 dye (Life Technologies) conjugated to POPE and 103 an Alexa-Fluor® 488 dye conjugated to DPoPE. M21 cells were treated with these lipids and assessed by quantitative flow cytometry using calibration beads to determine the number of fluorophores per cell. The results are illustrated in FIG. 5. All three lipids showed robust insertion into cell membranes with copy numbers around 3×10⁶.

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US09861702B2. Lipid-conjugated rhamnose for immune system recruitment and oncotherapy (2018-01-09). WISCONSIN ALUMNI RESEARCH FOUNDATION [US]. Inventors: Laura L. Kiessling [US], Rachael T. C. Sheridan [US], Jonathan Hudon [US].

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Immunohistochemical Analysis of Testis Graft

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For immunohistochemical staining, tissue sections of the graft (both experimental and negative control) were stained with hematoxylin and eosin. A genitourinary pathologist (who was blinded to the samples) independently verified the presence of LCSs under 10x and 60x magnification. To confirm the presence of different cell types, fluorescence staining was performed on the three testis biopsies that were used for flow cytometry using (1) antibody against B3HSD (sc-30820) followed by Alexa Fluor 488 dye (Thermo Fisher Scientific); (2) anti-alpha SMA antibody (AB5694) followed by Alexa Fluor 488 dye; (6) anti-smooth muscle Myosin heavy chain 11 mAb (SMHC11) followed by Alexa Fluor 568 dye; (7) anti-Vimentin mAb (ab45939) followed by Alexa Fluor 568 dye. All samples were assessed under a fluorescence microscope (Leica Microsystem, Wetzlar, Germany) at 60x. Images were acquired using MetaMorph version 4.6 (Molecular Devices, Sunnyvale, CA, USA) (information on antibodies can be found in Resource Table).

Arora H., Qureshi R., Khodamoradi K., Seetharam D., Parmar M., Van Booven D.J., Issa I.C., Sackstein R., Lamb D., Hare J.M, & Ramasamy R. (2022). Leptin secreted from testicular microenvironment modulates hedgehog signaling to augment the endogenous function of Leydig cells. Cell Death & Disease, 13(3), 208.

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Visualization of Intracellular and Surface-Exposed DENV2 NS1 Protein

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Mock or DENV2-infected human immortalized hepatocyte-like cells (imHC) were used to observe intracellular and surface-exposed NS1 protein. For intracellular NS1 staining, cells were fixed and permeabilized with 2% formaldehyde and 0.1% triton X-100, respectively. Human anti-NS1 mAbs were added to the cells, followed by rabbit anti-human IgG antibody conjugated with Alexa Fluor 488 dye (Invitrogen) at a 1:500 dilution. For surface-exposed NS1 staining, live cells were detected with antibodies as described above. Propidium iodide (PI) was added immediately before flow cytometry to exclude dead cells. Flow cytometry of processed cells was performed on BD FACSCalibur™ Flow Cytometer (BD Bioscience, USA).
The binding of NS1 to cell surfaces was determined as described previously [9 (link)]. Briefly, imHC cells were removed from tissue culture plates with an EDTA solution (8mM EDTA in plain DMEM-F12). Cells (2 x 10⁵) in suspension were incubated on ice for 1 h with purified DENV2 NS1 (1 μg). After washing with serum-free medium, human anti-NS1 antibodies were added to the cells and incubated on ice for 1 h. Bound primary antibodies were detected by a 1:500 diluted goat anti-human IgG conjugated with Alexa Fluor 488 dye (Invitrogen). PI was added before detection.

Kraivong R., Traewachiwiphak S., Nilchan N., Tangthawornchaikul N., Pornmun N., Poraha R., Sriruksa K., Limpitikul W., Avirutnan P., Malasit P, & Puttikhunt C. (2022). Cross-reactive antibodies targeting surface-exposed non-structural protein 1 (NS1) of dengue virus-infected cells recognize epitopes on the spaghetti loop of the β-ladder domain. PLoS ONE, 17(5), e0266136.

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Placental Apoptosis in Obesity

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Paraformaldehyde (PFA) post-fixed hemi-sectioned placentae (Lean, N = 13; VSG N = 9; Obese, N= 9) were subjected to standard paraffin-embedding, and then sectioned at 5 μm on to glass slides and processed for TUNEL staining with Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor® 488 dye (#C10617, Molecular Probes, Inc., Eugene, OR), according to the manufacturer’s specifications.

Spann R.A., Lawson W.J., Bidwell GL I.I.I., Zamarripa C.A., Maranon R.O., Bandyopadhyay S., Taylor E.R., Reckelhoff J.F., Garrett M.R, & Grayson B.E. (2018). Rodent Vertical Sleeve Gastrectomy Alters Maternal Immune Health and Feto-placental Development. Clinical science (London, England : 1979), 132(2), 295-312.

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Neuronal Autophagy Regulation in HIV-Morphine Coexposure

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Primary human neurons were purchased from ScienCell Research Laboratories (catalog number 1520) and cultured according to the manufacturer’s instructions. Cells were treated with supernatants from uninfected or HIV-1-infected (30 pg/mL HIV-1 p24) microglia and 500 nM morphine sulfate (Sigma-Aldrich; St. Louis, MO, USA; catalog number M8777) alone or in combination for 24 h and fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 10% milk/0.1% goat serum, and immunolabeled. Primary antibodies were anti-p62/SQSTM1 (Novus Biologicals; catalog number NBP1-48320) and anti-MAP2 (Abcam; catalog number ab5392), both used at a 1:100 dilution. Immunoreactivity was visualized with secondary antibodies from Molecular Probes conjugated to Alexa Fluor 594 dye (catalog number A11037) for p62/SQSTM1 and Alexa Fluor 488 dye (catalog number A11039) for MAP2, both used at a 1:500 dilution. DAPI staining was used to label cell nuclei.

Dever S.M., Rodriguez M., Lapierre J., Costin B.N, & El-Hage N. (2015). Differing roles of autophagy in HIV-associated neurocognitive impairment and encephalitis with implications for morphine co-exposure. Frontiers in Microbiology, 6, 653.

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Immunoblotting and Immunofluorescence Assays

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Anti-ERK1/2, anti-phospho-ERK1/2 and anti-phospho-Akt polyclonal antibodies were purchased from Cell Signaling (Beverly, MA). Anti-β-actin monoclonal antibody was from Sigma (St Louis, MO). Anti-hCXCR4 monoclonal antibody for immuno-fluorescent staining was from R&D Systems (Minneapolis, MN). Recombinant human SDF-1α was purchased from PeproTech EC Ltd. (London, UK). Alexafluor- 488 dye was obtained from Molecular Probes. LY2624587 is a recombinant antibody and available through a material transfer agreement with Eli Lilly and Company.

Peng S.B., Zhang X., Paul D., Kays L.M., Ye M., Vaillancourt P., Dowless M., Stancato L.F., Stewart J., Uhlik M.T., Long H., Chu S, & Obungu V.H. (2016). Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies. PLoS ONE, 11(3), e0150585.

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Dextran Amine Tracing of Inner Ear Afferents

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Dextran amine dye injections into ears transplanted adjacent to the spinal cord were used to evaluate inner ear afferent projection in the CNS. Entry points of inner ear afferents into the spinal cord as well as their projections into the hindbrain were evaluated using Texas red, tetramethylrhodamine, Alexa Fluor 647, and Alexa Fluor 488 dye (Molecular Probes). A small incision was made into the transplanted ear of anesthetized animals (0.02% Benzocaine) and a recrystallized drop of the labeling dye on a tungsten needle was inserted (Fritzsch, 1993 (link)). Care was taken to fill the ear entirely with the dye. Animals were washed in 0.1X MMR three times in succession and kept in a dish containing 0.1X MMR for 2–3 hr. Afterwards, the embryos were reanesthetized in 0.02% Benzocaine and fixed in 4.0% PFA. After fixation, the brain and spinal cord was dissected out and the specimens were mounted in glycerol for imaging on a TCS SP5 Multiphoton confocal microscope using appropriate excitation/emission filter settings. Dextran amine tracing served to verify lipophilic dye tracing as it is not known to diffuse transcellularly.

Gordy C., Straka H., Houston D.W., Fritzsch B, & Elliott K.L. (2018). Caudal Transplantation of Ears Provides Insights into Inner Ear Afferent Pathfinding Properties. Developmental neurobiology, 78(11), 1064-1080.

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Immunostaining of Autophagic Markers in Brain Tissue

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Frozen brain tissue from the NNTC Gene Array Project subjects was sectioned to 5 micron thickness, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 10% milk/0.1% goat serum, and immunolabeled. Primary antibodies used were anti-p62/SQSTM1 (catalog number NBP1-48320) at a 1:100 dilution and anti-Beclin 1 (catalog number NB500-249), anti-LC3B (catalog number NB600-1384), anti-ATG5 (catalog number NB110-53818), and anti-LAMP1 (catalog number NB120-19294) at a 1:400 dilution from Novus Biologicals (Littleton, CO, USA); anti-APG7/ATG7 (Santa Cruz Biotechnology; Santa Cruz, CA, USA; catalog number sc-33211) at a 1:100 dilution; and anti-Iba1 (Abcam; Cambridge, MA, USA; catalog number ab5076) at a 1:100 dilution. Immunoreactivity was visualized with secondary antibodies from Molecular Probes (Carlsbad, CA, USA) conjugated to Alexa Fluor 488 dye (catalog number A11055) for Iba1 and Alexa Fluor 594 dye (catalog number A11037) for autophagy proteins and related markers, both used at a 1:200 dilution. DAPI staining was used to label cell nuclei.

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Indirect Immunofluorescence Staining Protocol

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Indirect immunofluorescence staining was performed as described previously (33 (link)–34 (link), 37 (link)). Background fluorescence was determined by applying the secondary conjugated antibody alone and by replacement of the primary antibody with non-immune serum. Slides were successively stained with β-catenin antibody (clone 14; 1:100; BD Biosciences, San Jose, CA), followed by incubation with secondary antibodies labeled with Alexa Fluor 488 Dye (Molecular Probes Life Technologies). Images were collected using a confocal laser scanning system (LSM 510, Karl Zeiss, Gottingen, Germany).

Ono M., Yin P., Navarro A., Moravek M.B., Coon V J.S., Druschitz S.A., Gottardi C.J, & Bulun S.E. (2014). Inhibition of canonical WNT signaling attenuates human leiomyoma cell growth. Fertility and Sterility, 101(5), 1441-1449.e1.

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Immunofluorescence Microscopy Protocol

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For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde for 30 min. Subsequently, they were permeabilized and blocked for 30 min with 0.1% Triton X‐100 (#T8787, Sigma‐Aldrich), 10% goat serum (#16210072, Gibco^®, Life Technologies), and 1% BSA (#A9647, Sigma‐Aldrich) in PBS (#20012‐019, Gibco^®, Life Technologies). When AGO1 antibodies were used for staining, the blocking buffer was modified to use donkey serum (D9663‐10ML) instead of goat serum. Thereafter, the cells were incubated with primary antibodies (1:100 dilution) in the same buffer at desired dilution overnight at 4°C. Secondary anti‐rabbit, anti‐goat, or anti‐mouse antibodies labeled either with Alexa Fluor^® 488 dye (green), Alexa Fluor^® 568 dye (orange), Alexa Fluor^® 594 dye (red), or Alexa Fluor^® 647 dye (far red) fluorochromes (Molecular Probes) were used at 1:500 dilutions. The cells were mounted on a glass slide with VECTASHIELD DAPI (Vector Laboratories), and cells were mostly observed and documented with a ZEISS point scanning confocal LSM 700/LSM 800 inverted microscopes with a PLAN APO 40× (NA = 1.3) and 63× (NA = 1.4) oil‐immersion objectives. Laser settings and digital gain settings were set so as to avoid any saturated pixel in the resulting capture. Z‐stack images were captured wherever mentioned in text.

Ghosh S., Guimaraes J.C., Lanzafame M., Schmidt A., Syed A.P., Dimitriades B., Börsch A., Ghosh S., Mittal N., Montavon T., Correia A.L., Danner J., Meister G., Terracciano L.M., Pfeffer S., Piscuoglio S, & Zavolan M. (2020). Prevention of dsRNA‐induced interferon signaling by AGO1x is linked to breast cancer cell proliferation. The EMBO Journal, 39(18), e103922.

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