Alexa fluor 488

Manufactured by Abcam

Sourced in United Kingdom, United States, Germany, Japan, China, Canada, France

Alexa Fluor 488 is a fluorescent dye used in various biological applications. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, making it suitable for detection in the green fluorescence range. This dye is commonly used for labeling proteins, cells, and other biomolecules for imaging and analysis purposes.

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Lab products found in correlation

Alexa fluor 647, by Abcam (56 mentions) Alexa fluor 594, by Abcam (51 mentions) Ab150077, by Abcam (24 mentions) Alexa fluor 555, by Abcam (23 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (23 mentions) Triton x 100, by Merck Group (17 mentions) Alexa fluor 568, by Abcam (15 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (15 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (14 mentions) Fluorescence microscope, by Olympus (12 mentions) Bovine serum albumin (bsa), by Merck Group (12 mentions) Alexa fluor 594, by Thermo Fisher Scientific (10 mentions) Lsm 710 confocal microscope, by Zeiss (9 mentions) Paraformaldehyde (pfa), by Merck Group (8 mentions) Triton x 100, by Beyotime (8 mentions) Fluorescence microscope, by Leica camera (7 mentions) Normal goat serum (ngs), by Thermo Fisher Scientific (7 mentions) Lsm 700, by Zeiss (7 mentions) Lsm 880 confocal microscope, by Zeiss (7 mentions) Mitotracker red, by Thermo Fisher Scientific (7 mentions)

494 protocols using alexa fluor 488

Immunofluorescence Staining of Retinal Cells

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The frozen sections were blocked with 5% BSA/0.3% Triton X-100 at room temperature for 2 h, and then incubated with primary antibodies against CD45 (1:200; BD Pharmingen, CA, USA), IBA1 (1:500; Wako Pure Chemical Industries, Japan) or GFAP Cy3 Conjugate (1:500; Sigma-Aldrich, USA) at 4 °C overnight. After washing three times with PBS, sections were incubated with secondary antibodies conjugated with Alexa Fluor 488 (1:500; Abcam) or Alexa Fluor 647 (1:500; Abcam) for 2 h at room temperature, stained with DAPI, and photographed using a confocal microscope (Zeiss LSM800 or Zeiss LSM880).
For flat-mounted retina, the eyeballs (n = 6/group) were enucleated 7 days after OAB and fixed in 4% paraformaldehyde for 50 min. The whole retinas were carefully prepared and permeabilized with 0.3% Triton X-100 and blocked with 5% BSA overnight. Next, they were incubated with primary antibodies against Brn3a (1:500; Santa Cruz Biotechnology) or IBA1(1:500; Wako Pure Chemical Industries) for 24 h and washed in PBS, and then incubated with secondary antibody conjugated with Alexa Fluor 488 (1:500; Abcam). After three more PBS washes, DAPI was used to stain sections for 10 min. Finally, retinas were mounted with anti-fade mounting medium. All images were collected by a confocal microscope (Zeiss LSM800 or Zeiss LSM880).

Huang Y., Lin L., Yang Y., Duan F., Yuan M., Lou B, & Lin X. (2022). Effect of Tauroursodeoxycholic Acid on Inflammation after Ocular Alkali Burn. International Journal of Molecular Sciences, 23(19), 11717.

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Immunofluorescence Analysis of TDP-43, SRSF1, SRSF2, and NCL

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Slides with NSC34 cells were fixed and permeabilised in 4% PFA/0.2% Triton X-100 at RT for 20 min. Slides were incubated with rabbit polyclonal anti-TDP-43 (1 in 200, Proteintech, 10782-2-AP), rabbit polyclonal anti-SRSF1 (1 in 200, Abcam ab38017), mouse monoclonal anti-SRSF2 (1 in 200, Abcam ab11826), or rabbit polyclonal anti-NCL (1:200; Proteintech; 10556-1-AP) in 2% BSA in PBS at RT for 1 h. Slides were washed three times in PBS, then incubated with goat anti-rabbit IgG H&L (1 in 1000; Alexa Fluor® 488; Abcam) or goat anti-mouse IgG H&L (1 in 1000; Alexa Fluor® 488; Abcam) in 2% BSA in PBS at RT for 1 h. Formalin fixed paraffin-embedded tissue sections were deparaffinized and mouse anti-NCL (1 in 150 in 5% BSA; Abcam; ab136649) stained slides were first antigen retrieved by heat in trisodium citrate pH 6.5 for 20 min, then completed as for ICC. Slides of NSC34 and tissue sections were mounted with mounting medium containing DAPI (Vector Labs Inc.).

Stopford M.J., Higginbottom A., Hautbergue G.M., Cooper-Knock J., Mulcahy P.J., De Vos K.J., Renton A.E., Pliner H., Calvo A., Chio A., Traynor B.J., Azzouz M., Heath P.R., Kirby J, & Shaw P.J. (2017). C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten. Human Molecular Genetics, 26(6), 1133-1145.

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Microglia Activation and Neuroinflammation Study

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The microglial activator lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA, L2630). The antibodies used for western blotting in this study included anti-GAPDH (Abcam, Rabbit, ab181602), anti-CD9 (Abcam, Rabbit, ab195422), anti-CD63 (Abcam, Rabbit, ab217345), anti-CD81 (Abcam, Rabbit, ab109201), anti-calnexin (Abcam, Rabbit, ab22595), anti-INOS (Abcam, Mouse, ab49999), anti-C3 (Abcam, Rabbit, ab97462), anti-MYH9 (Abcam, Mouse, ab55456), anti-p-PI3K (CST, Rabbit, 17366), anti-PI3K (CST, Rabbit, 4292), anti-p-AKT (CST, Rabbit, 4060), anti-AKT (CST, Mouse, 2920), anti-p-P65 (CST, Rabbit, 3033), and anti-P65 (CST, Rabbit, 8242). The antibodies used for immunofluorescence were anti-NEUN (Abcam, Rabbit, ab177487), anti-MAP2 (Abcam, Mouse, ab11267), anti-INOS (Abcam, Mouse, ab49999), anti-C3 (Abcam, Rabbit, ab97462), anti-IBA1 (Abcam, Rabbit, ab178846), anti-GFAP (Abcam, Mouse, ab10062), and anti-NF200 (Abcam, Rabbit, ab204893). and secondary antibodies were goat anti-mouse Alexa Fluor 488 (Abcam, Goat, ab150113), goat anti-rabbit Alexa Fluor 594 (Abcam, Goat, ab150088), goat anti-mouse Alexa Fluor 594 (Abcam, Goat, ab150120), goat anti-rabbit Alexa Fluor 488 (Abcam, Goat, ab150077). ELISA kits were TNF-α (R&D, MTA00B), IL-1α (R&D, MLA00), IL-1β (R&D, MLB00C), IL-6 (R&D, M6000B), and C1q (Hycult Biotech, HK211).

Jiang D., Gong F., Ge X., Lv C., Huang C., Feng S., Zhou Z., Rong Y., Wang J., Ji C., Chen J., Zhao W., Fan J., Liu W, & Cai W. (2020). Neuron-derived exosomes-transmitted miR-124-3p protect traumatically injured spinal cord by suppressing the activation of neurotoxic microglia and astrocytes. Journal of Nanobiotechnology, 18, 105.

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Characterization of Neural Cells in NBA Cultures

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Cells in NBA cultures were validated by a double immunostaining assay using glial and neuronal specific markers. Cells were first fixed with 4% PFA for 25 min and then washed twice in PBS (1X) for 5 min. DCs were permeabilized in PBST (0.1% Triton-X in PBS 1X) 3 times for 10 min each then blocked (5% FBS in PBST 1X) for 30 min at room temperature. DCs were then rinsed with PBST 3 times for 10 min each, followed by subsequent midnight incubation with primary antibodies against GFAP (mouse anti-GFAP, Abcam, 1/1,000), Tuj1 (rabbit anti-mouse PAb to beta-Tubulin III, Abcam, 1/2,000) and Neuronal Nuclei NeuN (rabbit anti-mouse PAb to NeuN, Abcam, 1/1,000). Appropriate secondary antibodies (donkey anti-mouse PAb, Alexa-fluor 488, 1/1,000 and goat anti-rabbit, Alexa Fluor 488, 1/1,000, Abcam) were added for 1h at RT followed by washing the cells in PBST (3x, 10 min each). Hoechst was then added for 5 min to counter stain the DCs nuclei which were then washed in PBST (2x for 10 min each). Finally, DCs were mounted on microscopic slides and revealed by a fluorescent microscope (Axio Observer Inverted Microscope, Carl Zeiss, Germany).

Nasser M., Ballout N., Mantash S., Bejjani F., Najdi F., Ramadan N., Soueid J., Zibara K, & Kobeissy F. (2018). Transplantation of Embryonic Neural Stem Cells and Differentiated Cells in a Controlled Cortical Impact (CCI) Model of Adult Mouse Somatosensory Cortex. Frontiers in Neurology, 9, 895.

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Neural Stem Cell Autophagy Regulation

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NSCs culture medium was provided by Chi Scientific (Jiangsu, China). Antibodies to HIF-1α (ab1), bFGF (ab208687), LC3-II (ab192890), Beclin 1 (ab217179), P62 (ab109012), NeuN (ab177487), GAP43 (ab75810), GFAP (ab33922), and NF200 (ab4680) were supplied by Abcam (Cambridge, Britain). Goat anti-rabbit IgG- conjugated with Alexa Fluor594 (red, ab150080) or Alexa-Fluor488 (green, ab150077) was from Abcam (Cambridge, Britain). Goat anti-mouse IgG-conjugated with Alexa Fluor594 (red, ab150116) or Alexa-Fluor488 (green, ab150113) was from Abcam (Cambridge, Britain). Goat anti-chicken IgY-conjugated with Alexa Fluor594 (red, ab150172) was from Abcam (Cambridge, Britain). An enhanced chemiluminescence (ECL) kit was provided by Bio-Rad (Hercules, CA, USA). DAPI (4′,6-diamidino-2-phenylindole), a fluorescent agent for cell nuclear staining was from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (RAPA) autophagy inducer was obtained from Cell Signaling Technology (Danvers, MA, United States), and 3-Methyladenine (3-MA) from Sigma-Aldrich (St. Louis, MO, USA).

Zhu S., Ying Y., Ye J., Chen M., Wu Q., Dou H., Ni W., Xu H, & Xu J. (2021). AAV2-mediated and hypoxia response element-directed expression of bFGF in neural stem cells showed therapeutic effects on spinal cord injury in rats. Cell Death & Disease, 12(3), 274.

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Immunofluorescence Analysis of Skin Tissue

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After deparaffinization, antigen retrieval, and permeabilization, the 3-μm skin tissue sections were incubated with 3% H₂O₂ and blocked using 5% goat serum. Processed sections were performed with primary antibody against MIF (1:1000, Abcam, USA), CD74 (1:500, Santa Cruz, CA, USA), Melan-A (1:500), E-Cadherin (1:1000, Abcam, USA), arachidonate 15-lipoxygenase (ALOX15, 1:1000, Abcam, USA) and 8-OHdG (1:1000, Abcam, USA) at 4°C overnight, respectively. After washing with PBS for 3 times, the goat anti-rabbit antibody Alexa Fluor^® 488 (1:1000, Abcam), goat anti-mouse antibody Alexa Fluor^® 488 (1:1000, Abcam), and goat anti-rabbit antibody Alexa Fluor^® 555 (1:1000, Abcam) were dropped into the section for incubation for 1 h at room temperature, respectively. DAPI (1:1000) was added in the sections for 10 min. After the above steps were completed, an appropriate amount of anti-fluorescence quenching sealing solution containing DAPI (Abcam, USA) was dropped onto the slices for sealing and drying. Fluorescence detection was conducted under the Pannoramic MIDI (3D HISTEC, Hungary).

Tang N., Liu X.T., Lin X.L., Yang W.X., Li Q.L., Wang G.E, & Wu Y.H. (2024). Erzhiwan Ameliorates Restraint Stress- and Monobenzone-Induced Depigmentation in Mice by Inhibiting Macrophage Migration Inhibitory Factor and 8-Hydroxy-2-Deoxyguanosine. Clinical, Cosmetic and Investigational Dermatology, 17, 147-158.

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Immunofluorescence Staining of Rat Heart

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The hearts of rats were carefully excised, fixed in 4% paraformaldehyde for 24 hours, dehydrated with 30% sucrose, and embedded in OCT (Sakura Finetek, Inc., Torrance, CA, USA). Short axis-oriented cryosections of 5-µm thick were cut for immunofluorescence staining. Slides were blocked with normal goat serum for 1 hour, followed by incubation with primary antibodies overnight. Anti-rat CD18 antibody (Abcam, Cambridge, UK) was used for detection of leukocytes, anti-rat CD68 antibody (Abcam) for macrophages, anti-rat CD31 antibody (Abcam) for blood vessels, and anti-rat laminin antibody (Abcam) for basal membranes. Goat-anti-rabbit Alexa Fluor^® 488 (Abcam) or goat-anti-mouse Alexa Fluor^® 488 (Abcam) was used as the second antibody. Fluorescently labeled antibodies and Cy5.5 were tracked by a laser scanning confocal microscope (TCS SP5, Leica, Wetzlar, Germany).

Zheng Y., Zhang H., Hu Y., Bai L, & Xue J. (2018). MnO nanoparticles with potential application in magnetic resonance imaging and drug delivery for myocardial infarction. International Journal of Nanomedicine, 13, 6177-6188.

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Flow Cytometric Analysis of Cardiomyocytes

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EBs from D14 cultures were harvested and washed in ice-cold PBS. Cells were trypsinized for 2 min. Single-cell suspensions were created and adjusted to a concentration of 1 × 10⁶ cells/mL in ice-cold PBS with 1% FBS. Cells were stained with fixable viability dye eFluor 780 (1:1,000; Life Technologies) for 15 min at 4°C. After washing three times with ice-cold PBS, cells were fixed in 3.7% formaldehyde in PBS for 15 min, followed by blocking with 1% FBS in PBS 1× for 30 min at 4°C. Anti-cardiac troponin I antibody (Alexa Fluor 488) (Abcam catalog no. 196384) was diluted 1:50 in 1% FBS in PBS and incubated with cells for 30 min at 4°C following washing three times with PBS. Isotype control antibody was rabbit monoclonal IgG Alexa Fluor 488 (Abcam catalog no. 199091) used at the same concentration and conditions as the primary antibody. Non-viable cells were excluded from analysis. An LSR II flow cytometer was used for cell sorting, where at least 10,000 events of viable cells were collected. FlowJo V10 (Trestar) was used for data analysis and gating of positive populations was performed based on appropriate isotype-matched antibody control. Different repeats were analyzed using the same settings for gating.

Karimzadeh F, & Opas M. (2017). Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells. Stem Cell Reports, 8(5), 1299-1311.

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Immunofluorescence Staining of Osteocalcin

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After 2 days of culture, the cells were washed twice with PBS and fixed with 4% Paraformaldehyde (PFA, Sigma–Aldrich) for 10 min at room temperature followed by three PBS washes. Plates were then sealed with parafilm and stored in the fridge until immunofluorescence. Cells were permeabilized with PBS containing 0.1% Triton (Sigma–Aldrich; TPBS) for 10 min and washed in PBS three times before being incubated with a blocking solution containing 10% donkey serum (Merck) in TPBS for 30 min. The blocking solution was then aspirated and the samples were washed three times in TPBS washes. The cells were then incubated with osteocalcin (1:200, Abcam, Cambridge, UK) in 10% donkey serum and 0.1% TPBS overnight at 4 °C. After 24 h, the primary antibody solution was decanted and the cells were washed three times with PBS and incubated with the secondary antibody Alexa Fluor 488 (1:500, Abcam) in 5% donkey serum and 0.2% TPBS for 1.5 h at room temperature. Following this, cells were incubated for 5 min with Hoechst (1:5000, Life Technologies, Scoresby, Australia) and three PBS washes were performed prior to mounting the coverslips on a glass slide. Cells were then visualized under a fluorescence microscope (Inverted Laboratory Microscope, Leica DM IL LED, Wetzlar, Germany).

Pistone A., Iannazzo D., Celesti C., Piperopoulos E., Ashok D., Cembran A., Tricoli A, & Nisbet D. (2019). Engineering of Chitosan-Hydroxyapatite-Magnetite Hierarchical Scaffolds for Guided Bone Growth. Materials, 12(14), 2321.

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Immunohistochemical Characterization of Tumor Cells

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Tumor tissues in the PDX assays and MiniPDX assays were fixed in buffered 10% formalin and routinely stained with hematoxylin and eosin (H&E) and examined by a certified pathologist.
For immunofluorescence studies, cellularized tumor cells (2 × 10⁴ cells, 200 L) were cytospun onto a slide, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and then blocked with 5% normal goat serum for 1 h at room temperature. The cells were then divided into three fractions and incubated with primary mouse monoclonal antibodies at 4 °C overnight against the following proteins: pan-cytokeratin, indicating carcinoma components [27 (link), 28 (link)] (1:200, AE1/AE3, sc-81714, Santa Cruz Biotechnology, Santa Cruz, CA, US), E-cadherin, generally found in gastric adenocarcinomas [29 (link)] (1:50, HECD-1, ab1416, Abcam, Cambridge, UK), and MG7, a marker of gastric cancer [30 (link)] (1:300, NOTA-MG7) [30 (link)]. Subsequently, the cells were probed with secondary antibody donkey anti-mouse IgG H&L (Alexa Fluor^® 488) (1:200, ab150105, Abcam). Finally, the cells were mounted with DAPI-containing mounting medium (S36973, Thermo Fisher, MA, US). Images were captured with a fluorescence microscope (Leica, Germany) with Leica Application Suite V4 software and edited with Photoshop (Adobe, US).

Zhang F., Wang W., Long Y., Liu H., Cheng J., Guo L., Li R., Meng C., Yu S., Zhao Q., Lu S., Wang L., Wang H, & Wen D. (2018). Characterization of drug responses of mini patient-derived xenografts in mice for predicting cancer patient clinical therapeutic response. Cancer Communications, 38, 60.

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