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Alexa fluor 594

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Alexa Fluor 594 is a fluorescent dye developed by Thermo Fisher Scientific. It is designed to emit light in the red-orange region of the visible spectrum when excited with appropriate wavelengths of light. The dye can be used in various biological and biochemical applications that require a fluorescent label.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (1165 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (120 mentions) Alexa fluor 647, by Thermo Fisher Scientific (79 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (69 mentions) Hoechst 33342, by Thermo Fisher Scientific (68 mentions) Triton x 100, by Merck Group (59 mentions) Bovine serum albumin (bsa), by Merck Group (43 mentions) Vectashield, by Vector Laboratories (38 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (36 mentions) Paraformaldehyde (pfa), by Merck Group (29 mentions) Lsm 710 confocal microscope, by Zeiss (29 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (26 mentions) Lsm 780 confocal microscope, by Zeiss (23 mentions) Lsm 710, by Zeiss (22 mentions) Vectashield mounting medium, by Vector Laboratories (22 mentions) Axio observer z1, by Zeiss (21 mentions) Lsm 700 confocal microscope, by Zeiss (20 mentions) Lsm 780, by Zeiss (19 mentions) Alexa fluor 568, by Thermo Fisher Scientific (17 mentions) Vectashield with dapi, by Vector Laboratories (16 mentions)

2 035 protocols using alexa fluor 594

1

Immunofluorescent Staining of Aortic Tissues

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For immunofluorescent staining of the aortic tissue sections, we used sodium citrate (0.01 M, pH 6.0) for antigen retrieval. Sections were then blocked for 30 min with 5% BSA in PBS at room temperature. The following diluted primary antibodies: mouse anti-BKCa α (1:200, Abcam, Cambridge, MA, UK), rabbit anti-BKCa β (1:1000, Abcam, Cambridge, MA, UK), rabbit anti-Kir6.1 (1:200, Santa Cruz, CA, USA), mouse anti-Kir6.2 (1:50, Santa Cruz, CA, USA), rabbit anti-Cav1.2 (1:100, Santa Cruz, CA, USA), goat anti-Cav1.3 (1:200, Santa Cruz, CA, USA), were used to incubate sections overnight at 4 °C. Slides were then incubated at 37 °C with secondary antibodies Alexa Fluor 488 (donkey anti-goat, 1:400; Life Technologies), Alexa Fluor 488 (donkey anti-rabbit, 1:400; Life Technologies), Alexa Fluor 488 (donkey anti-mouse, 1:400; Life Technologies), Alexa Fluor 594 (donkey anti-rabbit, 1:400; Life Technologies), Alexa Fluor 594 (donkey anti-mouse, 1:400; Life Technologies) and Alexa Fluor 594 (donkey anti-goat, 1:400; Life Technologies) for 1 h. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to counterstain all sections. The slides were then mounted and viewed under a fluorescent microscope (Olympus BX53).
Zhao Y., Ge J., Li X., Guo Q., Zhu Y., Song J., Zhang L., Ding S., Yang X, & Li R. (2019). Vasodilatory effect of formaldehyde via the NO/cGMP pathway and the regulation of expression of KATP, BKCa and L-type Ca2+ channels. Toxicology letters, 312, 55-64.
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2

Immunofluorescence Staining of HeLa and LHCNM2 Cells

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HeLa cells were seeded on poly-d-lysine coated glass slides and fixed 48 h posttransfection. HeLa cells were washed once with PBS (Gibco) and fixed with 2% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. Cells were washed with PBS and PBS+ (PBS with 0.5% BSA and 0.15% glycine)34 (link),49 (link). Primary antibody incubation was performed overnight at 4 °C in a humid chamber and Alexa-conjugated dyes (Invitrogen, Carlsbad, CA, USA; Alexa Fluor 488 goat-anti-Rabbit, Alexa Fluor 488 donkey-anti-Rabbit, Alexa Fluor 594 goat-anti-Rabbit, Alexa Fluor 594 donkey-anti-Rabbit, Alexa Fluor 488 goat-anti-mouse, Alexa Fluor 594 goat-anti-mouse, and Alexa Fluor 594 chicken-anti-rat) were applied for 1.5 h at room temperature to visualize primary antibodies. Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) or Hoechst 33258 for 5 min, and samples were and embedded in glycerol (CitiFluor, Agar Scientific). LHCNM2 cells were grown on poly-l-lysine coated glass coverslip. 24 h after seeding, cells were transfected using lipofectamine 3000 according to manufacturer protocol. After 24 h transfection, cells were fixed with 3.7% formaldehyde for 9 min at room temperature, permeabilized with cold (−20 °C) acetone for 5 min at −20 °C. Cells were rinsed with PBS 1× and processed for immunostaining.
Meister-Broekema M., Freilich R., Jagadeesan C., Rauch J.N., Bengoechea R., Motley W.W., Kuiper E.F., Minoia M., Furtado G.V., van Waarde M.A., Bird S.J., Rebelo A., Zuchner S., Pytel P., Scherer S.S., Morelli F.F., Carra S., Weihl C.C., Bergink S., Gestwicki J.E, & Kampinga H.H. (2018). Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks. Nature Communications, 9, 5342.
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3

Pancreatic Islet Immunohistochemistry

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Pancreata were isolated, fixed with 10% buffered formalin, and embedded in paraffin. Sections were prepared and either stained with hematoxylin and eosin for visualization of islet morphology by light microscopy or subjected to immunohistochemistry. Nitrated proteins were detected with anti-nitrotyrosine antibody (Upstate) using the Histostatin-Plus Kit (Zymed Laboratories). Anti-insulin (1:200; Linco, 4011–01) and anti-glucagon (1:200; Linco, 4030–01F) antibodies were identified with anti–guinea pig Texas red (1:1000; Jackson ImmunoResearch, 106–075–003,) and with goat anti-rabbit Alexa Fluor 488 as the secondary antibody (1:1000; Invitrogen, A11008), respectively. Anti-PDX1 (1:200; Millipore, 07–696) and anti-4-hydroxynonenal (1:200; Abcam, ab46545) antibodies were identified with antirabbit Alexa Fluor 594 as the secondary antibody (1:1000; Invitrogen, A11037). Anti-MafA (1:100; Santa Cruz Biotechnology, sc-27140) antibody was identified with anti-goat Alexa Fluor 594 as the secondary antibody (1:1000; Invitrogen, A11058). In Fig. 6F, anti-proinsulin (1:200; HyTest Ltd., 2PR8) antibody was identified with anti-mouse Alexa Fluor 594 (1:1000; Invitrogen, A11005). In Fig. 7C and Supplementary Fig. 8, anti-insulin antibodies was identified with anti–guinea pig Alexa Fluor 488 (1:1000; Invitrogen, A11073).
Han J., Song B., Kim J., Kodali V.K., Pottekat A., Wang M., Hassler J., Wang S., Pennathur S., Back S.H., Katze M.G, & Kaufman R.J. (2015). Antioxidants Complement the Requirement for Protein Chaperone Function to Maintain β-Cell Function and Glucose Homeostasis. Diabetes, 64(8), 2892-2904.
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4

Antibodies for DNA Damage Response

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The primary antibodies used were as follows: mouse anti-FLAG antibody (Sigma-Aldrich; F1804), mouse anti-γH2AX (Cell Signaling; #9718) and (Millipore; #05-636), rabbit anti-53BP1 (Novus Biologicals; NB100-304), mouse anti-53BP1 (BD transduction laboratories; 612522), rabbit anti-MDC1 (Abcam; ab11169), goat anti-Rif1 (N-20) (Santa Cruz, sc-55979), rabbit anti-beta-tubulin (Abcam; ab6046), mouse anti-c-Myc (Santa Cruz; sc-40), rabbit anti-H2AX (Cell Signaling; #2595). Secondary antibodies for western blotting were as follows: anti-rabbit igG, HRP-linked (Cell Signaling; #7074), anti-mouse IgG, HRP-linked (Cell Signaling; #7076). Secondary antibodies for IF analysis from Invitrogen were as follows: Alexa Fluor 488 (Rabbit, A11034), Alexa Fluor 594 (Rabbit, A11037), Alexa Fluor 594 (Mouse, A11032), Alexa Fluor 594 (Goat, A11058), Alexa Fluor 647 (Rabbit, A21245) and Alexa Fluor 647 (Mouse, A21236). For experiments involving cell cycle analysis, the following antibodies were used; mouse anti-BRCA1 (D-9) (Santa Cruz; sc-6954), mouse anti-RPA32/RPA2 [9H8] (Abcam; ab2175), rabbit anti-Phospho-histone H3 (Ser10) (D2C8) (Cell Signaling, #3377) and rabbit anti-CyclinA (H-432) and (Santa Cruz; sc-751).
Leung J.W., Agarwal P., Canny M.D., Gong F., Robison A.D., Finkelstein I.J., Durocher D, & Miller K.M. (2014). Nucleosome Acidic Patch Promotes RNF168- and RING1B/BMI1-Dependent H2AX and H2A Ubiquitination and DNA Damage Signaling. PLoS Genetics, 10(3), e1004178.
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5

Microscopic Visualization and Quantification of DNA Damage and R-Loops

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Cells were grown onto poly-lysine (Sigma-Aldrich P7280) coated coverslips, fixed in 4% (v/v) formaldehyde and processed as previously described (Kantidakis et al., 2016 (link)). Briefly, the primary antibody, used in 1:1000 dilution was anti 53BP1 (Abcam, ab 36823) and the secondary antibody anti-rabbit Alexa Fluor 594 (Life Technologies). The coverslips were mounted using Vectashield Antifade Mounting Medium containing DAPI (Vector Laboratories, H-1200) and visualized using a Zeiss fluorescent microscope with a 63x/1.4 oil immersion and quantified with ImageJ.
For R-loop detection, cells were grown on coverslips, fixed and permeabilized in 100% ice cold methanol and acetone for 10 min and 1 min on ice, respectively, and processed as previously described (Sridhara et al., 2017 (link)). Briefly, the primary antibody, S9.6 was used in 1:500 dilution, and the secondary antibody anti-mouse Alexa Fluor 594 (Life Technologies). The coverslips were mounted using Vectashield Antifade Mounting Medium containing DAPI (Vector Laboratories, H-1200) and visualized using a Zeiss fluorescent microscope with a 63x/1.4 oil immersion and quantified with ImageJ.
Zatreanu D., Han Z., Mitter R., Tumini E., Williams H., Gregersen L., Dirac-Svejstrup A.B., Roma S., Stewart A., Aguilera A, & Svejstrup J.Q. (2019). Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability. Molecular Cell, 76(1), 57-69.e9.
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6

Immunofluorescence Staining Procedure

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The immunofluorescence experiments were performed using the indicated antibodies and according to standard procedures63 (link). Briefly, cells were fixed in 3% PFA-PBS for 20 min and permeabilized using PBS with 0.5% NP-40. Following blocking with PBS with 0.1% NP-40 and containing 10% FBS, the coverslips were incubated with the antibodies as indicated for two to three hours (anti-H2Aub at 1/5000 and anti-Myc antibody at 1/1000). Anti-mouse Alexa Fluor® 594, anti-mouse Alexa Fluor® 488, Anti-rabbit Alexa Fluor® 488, or anti-rabbit Alexa Fluor® 594 (Life Technologies) were used as secondary antibodies at 1/2000 and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using BX53 OLYMPUS microscope U-HGLGPS, XM10 digital monochrome camera and UPlan SApo 60 × /1,35 Oil objective. Images were processed using WCIF-ImageJ software (NIH).
Daou S., Barbour H., Ahmed O., Masclef L., Baril C., Sen Nkwe N., Tchelougou D., Uriarte M., Bonneil E., Ceccarelli D., Mashtalir N., Tanji M., Masson J.Y., Thibault P., Sicheri F., Yang H., Carbone M., Therrien M, & Affar E.B. (2018). Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1. Nature Communications, 9, 4385.
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7

Murine Macrophage Immunofluorescence Assay

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Murine specimens were fixed in 4% paraformaldehyde for 24 hours, dehydrated in 18% sucrose solution, mounted in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, California, USA), and sectioned at 5 μm. Heat mediated retrieval in Dako Target Retrieval Solution (Dako, Carpinteria, California, USA) was performed. The primary antibodies used were anti-F4/80 (MCA497R; AbD Serotec, Düsseldorf, Germany), anti-inducible nitric oxide synthase (iNOS) (ab15323–500; Abcam, Cambridge, Massachusetts, USA), and anti-arginase antibody (ab60176; Abcam). Secondary antibodies used were Alexa Fluor 488 (A-21208; Life Technologies, Grand Island, New York, USA) and Alexa Fluor 594 (A-21207; Life Technologies) or Alexa Fluor 594 (A-11058; Life Technologies). The nuclei were counterstained with DAPI (H-1200; Vector Labs). M1 macrophages were defined as F4/80+ iNOS+, while M2 macrophages were defined as F4/80+ Arg I+ cells.
Nowicki K.W., Hosaka K., Walch F.J., Scott E.W, & Hoh B.L. (2017). M1 macrophages are required for murine cerebral aneurysm formation. Journal of neurointerventional surgery, 10(1), 93-97.
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8

Alexa Fluor 594 Labeling Protocol

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The goat IgG isotype control and the goat IgG anti-SR-B1 were labeled with Alexa Fluor 594 using succinimidyl ester of Alexa Fluor 594 (Life Technologies) according to the manufacturer’s instructions.
Ganesan L.P., Mates J.M., Cheplowitz A.M., Avila C.L., Zimmerer J.M., Yao Z., Maiseyeu A., Rajaram M.V., Robinson J.M, & Anderson C.L. (2016). Scavenger receptor B1, the HDL receptor, is expressed abundantly in liver sinusoidal endothelial cells. Scientific Reports, 6, 20646.
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9

Histological Analysis of Mouse Intestine and Bone Marrow

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Mouse ileum and colon samples were frozen in OCT medium or formalin-fixed and embedded in paraffin. Sections of 5-7 μm in thickness on slides were stained with hematoxylin and eosin or incubated with marker-specific primary antibodies. Bone marrow smears on slides were air-dried, fixed in methanol, and stained with Wright-Giemsa dyes (Sigma) or incubated with marker-specific antibodies. For fluorescence labeling, the tissue sections and smears were incubated with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 or with streptavidin conjugated with Alexa Fluor 594 (Molecular Probes) and counterstained with Hoechst 33342 (Molecular Probes). Immunostained samples were analyzed by fluorescence microscopy.
Caballero-Franco C., Guma M., Choo M.K., Sano Y., Enzler T., Karin M., Mizoguchi A, & Park J.M. (2016). Epithelial control of gut-associated lymphoid tissue formation through p38α-dependent restraint of NF-κB signaling. Journal of immunology (Baltimore, Md. : 1950), 196(5), 2368-2376.
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10

Neutrophil Chikungunya Virus Interaction

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A total of 5 × 104 isolated neutrophils were attached on a slide coated with poly-D-lysine (Sigma-Aldrich) and incubated with CHIKV. After 4 h of incubation, the slides were washed with phosphate-buffered solution (PBS) and fixed with 4% paraformaldehyde for 30 min. The samples were blocked with a PBS/BSA 2% solution (Sigma-Aldrich) for 2 h at room temperature and incubated with primary antibodies against histone H3 citrulline R17+R2+R8 (ab5103, Abcam, 1:500), murine polyclonal anti-CHIKV antibodies (obtained from Dr. Figueiredo's lab, 1:100), and anti-Ly6G antibodies (16-9668-82, Invitrogen, 1:50) overnight at 4°C. After washing with PBS, anti-rabbit Alexa Fluor 488 (1:1000, Molecular Probes), anti-rat Alexa Fluor 594 (1:100, Molecular Probes), and/or anti-mouse Alexa Fluor 594 (1:200, Molecular Probes) were incubated for 2 h at room temperature. The slides were counterstained with DAPI (P36935, Molecular Probes), and the images were acquired with a Leica TCS SP5-AOBS microscope (Leica Microsystems, Mannheim, Germany).
Hiroki C.H., Toller-Kawahisa J.E., Fumagalli M.J., Colon D.F., Figueiredo L.T., Fonseca B.A., Franca R.F, & Cunha F.Q. (2020). Neutrophil Extracellular Traps Effectively Control Acute Chikungunya Virus Infection. Frontiers in Immunology, 10, 3108.
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