Triple-label immunofluorescence was performed as previously described [30 (link)] using Alexa Fluor™-488, -555, -647–conjugated secondary antibodies (Molecular Probes, Invitrogen), treated for autofluorescence with 0.1% Sudan Black solution, and coverslipped with ProLong-DAPI mounting medium (Molecular Probes). Digital images were obtained using an Olympus IX81 FluoView1000 microscope. Raw image z-stacks were analyzed using Imaris7.x software suite (Bitplane Scientific Software).
Alexa fluor 488 555 647 conjugated secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Alexa Fluor 488-/555-/647-conjugated secondary antibodies are fluorescent-labeled secondary antibodies used for detection and visualization in various immunoassays and imaging applications. These antibodies specifically bind to the Fc region of primary antibodies, allowing for the amplification and detection of target protein signals.
Automatically generated - may contain errors
Lab products found in correlation
α flag m2, by Merck Group (2 mentions)
Horseradish peroxidase conjugated anti mouse rabbit secondary antibodies for western blot analyses, by Dianova (2 mentions)
α myc 9e10, by Merck Group (2 mentions)
α β actin ac 15, by Merck Group (2 mentions)
Prolong dapi mounting medium, by Thermo Fisher Scientific (1 mentions)
Ix81 fluoview1000 microscope, by Olympus (1 mentions)
Tyramide signal amplification system, by PerkinElmer (1 mentions)
4 protocols using alexa fluor 488 555 647 conjugated secondary antibodies
1
Multicolor Immunofluorescence Imaging
2
Adult Eye Sectioning and Antibody Staining
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Protocols for adult eye sectioning and antibody staining have been described previously [6 (link), 19 (link)]. In all data analyses the R8 photoreceptors were individually identified to ensure that they were not mistaken as supernumerary R7s. Primary antibodies: rabbit anti-β galactosidase (Cappel); rabbit anti-GFP, mouse anti-GFP IgG2a (Molecular Probes); guinea pig anti-Runt (gift of J. Reinitz, Stony Brook University, NY, USA); rabbit anti-Runt (gift of Andrea Brand, Gurdon Institute, Cambridge, UK); guinea pig anti-Dpn (gift of James Skeath, Washington University School of Medicine, Washington, USA); mouse anti-Svp (gift of Y. Hiromi, National Institute of Genetics, Japan); rat anti-Elav, mouse anti-Cut (Developmental Studies Hybridoma Bank). Alexa Fluor 488, 555, 647 conjugated secondary antibodies were used (Molecular Probes). For amplification of the β-galactosidase staining, Donkey anti-rabbit HRP conjugated secondary antibodies were used. Amplification was conducted using Tyramide Signal Amplification (TSA) systems (PerkinElmer) following manufacturer’s protocol.
3
Antibody Usage in Immunofluorescence and Western Blot
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Monoclonal antibodies used for immunofluorescence and Western blot analyses were: α-IE1 63–27 [81 (link)], α-UL44 BS510 (kindly provided by B. Plachter, Mainz, Germany), α-MCP 28–4 [82 (link)], α-FLAG M2 (Sigma-Aldrich, Deisenhofen, Germany), α-Myc 9E10, α-β-actin AC-15 (Sigma-Aldrich, Deisenhofen, Germany), α-FEN1 B-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-phospho-Histone H2A.X (Ser139) (20E3) (Cell signaling technology, Danvers, MA, USA), α-E2F1 (KH95) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-BrdU MoBu-1 (Thermo Fisher Scientific, Waltham, MA, USA). The polyclonal antibodies α-phospho FEN1 (Ser187) (Thermo Fisher Scientific, Waltham, MA, USA) and α-p-Histone H2A.X (Ser 139) sc-101696 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and were used for Western blot analyses. Secondary antibodies used for immunofluorescence and Western blot analyses were: Alexa Fluor 488-/555-/647-conjugated secondary antibodies for indirect immunofluorescence experiments were purchased from Molecular Probes (Karlsruhe, Germany), horseradish peroxidase-conjugated anti-mouse/-rabbit secondary antibodies for Western blot analyses were obtained from Dianova (Hamburg, Germany).
4
Immunofluorescence and Western Blot Antibody Protocol
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Monoclonal antibodies used for immunofluorescence and Western blot analyses were: α-IE1 CH443 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-UL112/113 M23, α-UL44 BS510 (kindly provided by B. Plachter, Mainz, Germany), α-UL69 69–66, α-FLAG M2 (Sigma-Aldrich, Deisenhofen, Germany), α-Myc 9E10, α-β-actin AC-15 (Sigma-Aldrich), α-PML PG-M3 (Santa Cruz). Polyclonal antibodies used for immunofluorescence and Western blot analyses were: α-rhesus IE1 (a kind gift from M. Mach, Erlangen, Germany), α-PML #4 (a kind gift from P. Hemmerich, Jena, Germany), α-PML H238 (Santa Cruz), α-PML A301–167A (Bethyl Laboratories, Montgomery, TX, USA), α-PML A301–168A (Bethyl Laboratories), α-Sp100 #2 (a kind gift from P. Hemmerich, Jena, Germany), α-Sp100 GH3 (kindly provided by H. Will, Hamburg, Germany), α-hDaxx C-20 (Santa Cruz), α-ATRX H-300 (Santa Cruz). Secondary antibodies used for immunofluorescence and Western blot analyses were: Alexa Fluor 488-/555-/647-conjugated secondary antibodies for indirect immunofluorescence experiments were purchased from Molecular Probes (Karlsruhe, Germany), horseradish peroxidase-conjugated anti-mouse/-rabbit secondary antibodies for Western blot analyses were obtained from Dianova (Hamburg, Germany).
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