Alexafluor 595

Manufactured by Thermo Fisher Scientific

Sourced in United States

AlexaFluor 595 is a fluorescent dye that can be used for labeling biomolecules such as proteins, nucleic acids, and other biological molecules. It has an excitation maximum at 590 nm and an emission maximum at 617 nm, making it suitable for various fluorescence-based applications.

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Lab products found in correlation

Anti iba1 antibody, by Abcam (1 mentions) Anti cd31 antibody, by BD (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Fluoromount g, by Southern Biotech (1 mentions) Gelatin, by Bio-Rad (1 mentions) Occludin, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Bovine collagen 1, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Lsm 800, by Zeiss (1 mentions) Triton x 100, by Merck Group (1 mentions) Calretinin, by Abcam (1 mentions) Rabbit anti vimentin mab, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions) Recombinant tgf β1, by R&D Systems (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Rabbit anti trpv4, by Abcam (1 mentions) Flouromount, by Merck Group (1 mentions) Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (1 mentions) Ab74073, by Abcam (1 mentions)

6 protocols using alexafluor 595

Immunostaining Protocols for Mouse Microglia and Vasculature

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For mouse microglia immunostaining, eyes were enucleated and fixed in paraformaldehyde 4% for 1 hour. Subsequently, retinas and choroids were isolated, permeabilized, blocked and incubated with anti-Iba1 antibody (1/1000 dilution) (Abcam Ab178846) overnight, at room temperature. The next day, samples were washed, incubated with secondary antibody (1/200 dilution) (goat anti-rabbit AlexaFluor 595, Invitrogen A11012, USA) for 2 hours and washed again. Retinas and choroids were flat-mounted with Fluoromount-G (SouthernBiotech, The Netherlands) on glass-slides for microscopy imaging.
For mouse vascular immunostaining, eyes were enucleated and fixed in ethanol 70 % for 1 hour. Subsequently, choroids were isolated, permeabilized, blocked and incubated with anti-CD31 antibody (1/150 dilution) (Pharmingen 553370) overnight, at room temperature. The next day, samples were washed, incubated with secondary antibody (1/200 dilution) (goat anti-rat AlexaFluor 488, Invitrogen A11006) for 2 hours and washed again. Choroids were flat-mounted with Fluoromount-G (SouthernBiotech) on glass-slides for microscopy imaging.

Roblain Q., Louis T., Yip C., Baudin L., Struman I., Caolo V., Lambert V., Lecomte J., Noël A, & Heymans S. (2021). Intravitreal injection of anti-miRs against miR-142-3p reduces angiogenesis and microglia activation in a mouse model of laser-induced choroidal neovascularization. Aging (Albany NY), 13(9), 12359-12377.

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Visualizing Cytoskeleton and Tight Junctions in T. gondii-Challenged Cells

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T. gondii-challenged DCs were plated on coverslips coated with bovine collagen I (1 mg/ml, Life Technologies). After fixation (4% PFA, Sigma-Aldrich), cells were permeabilised (0.5% Triton X-100, Sigma-Aldrich) and stained with phalloidin Alexa Fluor 595 (Invitrogen). Micrographs were generated using a 63× objective (DMi8, Leica Microsystems). Primary MBECs were seeded on coverslips pre-coated with 0.1% gelatin (BioRad). Fixation and permeabilisation steps were carried out as for DCs, followed by blocking (5% FBS in PBS for 2 h). Cells were then incubated with primary antibodies to ZO-1 (ThermoFisher) and Occludin (ThermoFisher) ON at 4 °C at 1:500. Cells were then stained with Alexa Fluor 594-conjugated secondary antibodies (Invitrogen) and DAPI for 2 h, mounted and imaged by confocal microscopy (LSM 800, Zeiss).

Ross E.C., ten Hoeve A.L, & Barragan A. (2021). Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers. Cellular and Molecular Life Sciences, 78(12), 5197-5212.

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Investigating miR-29b-3p in EMT Regulation

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The oligonucleotides of miR-29b-3p mimics and negative control miRNA were synthesized by Thermo Fisher Scientific (Waltham, MA). The sequence of the oligonucleotides used for miR-29b-3pmimics.
5′-UAGCACCAUUUGAAAUCAGUGUU-3.
Lipofectamine RNAiMAX was purchased from Invitrogen (Carlsbad, CA). Rabbit mAbs to E-cadherin and calretinin, were purchased from Abcam (Cambridge, UK), Cell Signaling Technology (Danvers, MA). Recombinant- TGF-β1 was purchased from R&D systems (Minneapolis, MN). Rabbit anti-vimentin mAb and anti-rabbit Ig conjugated with AlexaFluor 488 or AlexaFluor 595® were from Invitrogen. DAPI was obtained from Dojindo (Kumamoto, Japan). Anti-integrin β1 mAb and RGD peptide were purchased from Cayman Chemical Co. (Ann Arbor, MI).
All methods were carried out in accordance with guidelines and regulations of the Declaration of Helsinki and all experimental protocols were approved by the Institutional Review Boards of Jichi Medical University (Approval number: RIN-A-19-161).

Kimura Y., Ohzawa H., Miyato H., Kaneko Y., Saito A., Takahashi K., Tojo M., Yamaguchi H., Kurashina K., Saito S., Hosoya Y., Lefor A.K., Sata N, & Kitayama J. (2022). MiR-29b may suppresses peritoneal metastases through inhibition of the mesothelial–mesenchymal transition (MMT) of human peritoneal mesothelial cells. Scientific Reports, 12, 205.

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Immunohistochemical Analysis of TRPV4, IL-1β, and Caspase-1 in Mouse Footpads

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Mice were perfused intracardially with 30 ml phosphate buffered saline, pH 7.4 (PBS), followed by 30 ml solution of 4% paraformaldehyde in PBS. Tissues were dissected out, post-fixed in 4% paraformaldehyde. Tissue blocks were further cryoprotected in 30% sucrose in PBS for 24-48 h and sectioned on a cryostat. Sections of footpads (10 μm)
were thaw-mounted onto slides. Sections were blocked with 5% normal goat serum (NGS; Jackson) in PBS/0.05% Tween20 (PBS-T), and incubated overnight with primary antibodies, rabbit anti-TRPV4 (1:300; Abcam), goat anti-IL1ß, (1:200; Santa Cruz Biotechnology Inc); rabbit anti-caspase-1(1:200; Biovision Research Products, CA). After washing, sections were incubated with secondary antibodies (AlexaFluor595 and AlexaFluor488-conjugated antibodies at 1:600; Invitrogen) for 2 h, rinsed, mounted, and cover-slipped with Flouromount (Sigma). Digital micrographs were obtained using a BX60
Olympus upright microscope equipped with high-res CCD camera and acquired with constant acquisition and exposure settings using ISEE software. Images were analyzed using imageJ open source software.

Moore C., Choi S., Moon G., Zhang J., Chen Y., & Liedtke W. (2021). Epidermal TRPV4 ion channels regulate UVB induced sunburn by triggering inflammmasome activation and ERK signaling.

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Quantification of FOXM1 Expression

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6000 cell/well, plated on 96-wells, were treated with drugs as indicated in figure legends. Then cells were fixed in 4% paraformaldehyde (20 min at RT), blocked by 0.2% PBS/BSA solution (5 min at RT) and incubated with primary anti-FOXM1 antibody for 1 h at 37 °C. After washes, cells were incubated with anti-rabbit Alexa Fluor 595 (Thermo Fisher Scientific, Waltham, USA) overnight at 4 °C. Then the cells were washed and incubated 15′ with 4′,6-diamidin-2-fenilindolo (DAPI) (Thermo Fisher Scientific, Waltham, USA). Representative images were taken at 40X magnification by Opera Phenix microscope (PerkinHelmer,Waltham, MA USA) and the positive cells are counted by Harmony software (PerkinHelmer,Waltham, MA, USA).

Roca M.S., Moccia T., Iannelli F., Testa C., Vitagliano C., Minopoli M., Camerlingo R., De Riso G., De Cecio R., Bruzzese F., Conte M., Altucci L., Di Gennaro E., Avallone A., Leone A, & Budillon A. (2022). HDAC class I inhibitor domatinostat sensitizes pancreatic cancer to chemotherapy by targeting cancer stem cell compartment via FOXM1 modulation. Journal of Experimental & Clinical Cancer Research : CR, 41, 83.

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Evaluation of SELENOK in Melanoma Cells

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Lysates from primary melanocytes were purchased from Sciencell Research Laboratories (Carlsbad, CA) and NCI-60 validated human melanoma cell lines obtained from the University of Hawaii Cancer Center included SK-Mel2, SK-Mel28, and MALME-3M. These cell lines were cultured in RPMI media with 10% fetal bovine serum and 1% Antibiotic-Antimycotic (all from GIBCO/Thermo Fisher). Primary antibodies for western blots included rabbit monoclonal anti-SELENOK (Epigemonics, Inc., custom antibody), anti-IP3R1 (Santa Cruz Biotechnology, sc-271197), anti-GAPDH (Santa Cruz Biotechnology, sc-47724), anti-Prom1 (Cell Signaling, 58605). Antibodies from Abcam included anti-TRP2 (ab74073), anti-calcineurin A and B (ab137335, ab154650), and anti-calmodulin (ab 105498, respectively). Western blot secondary antibodies were purchased from Li-Cor Technologies and immunofluorescence secondary Alexafluor595 antibody from Thermo. The transgene encoding full-length SELENOK in pcDNA3.1+ (Invitrogen) has been previously described [11 (link)].

Marciel M.P., Khadka V.S., Deng Y., Kilicaslan P., Pham A., Bertino P., Lee K., Chen S., Glibetic N., Hoffmann F.W., Matter M.L, & Hoffmann P.R. (2018). Selenoprotein K deficiency inhibits melanoma by reducing calcium flux required for tumor growth and metastasis. Oncotarget, 9(17), 13407-13422.

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