The following antibodies were used in this study: CB1 receptor (L15 pAb guinea pig, gift from Ken Mackie); synaptophysin (mouse, Synaptic Systems 101011); DARPP-32 (rabbit, Abcam ab40801); DAPI (Invitrogen D3571); 1C2 (mouse, Millipore MAB1574); GAPDH (mouse, Sigma 1405848; rabbit, Cell Signaling Technology 5174); Alexa Fluor 488/555/647 (goat/donkey, ThermoFisher); IRDye 680/800 (goat, LI-COR Biosciences); HRP-conjugated secondaries (goat, Cell Signaling Technology).
Alexa fluor 488 555 647
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488/555/647 are fluorescent dyes used in various biological applications. They are designed to emit light at specific wavelengths when excited, allowing for the detection and visualization of labeled molecules or structures. The core function of these dyes is to provide sensitive and photostable fluorescent labeling for a wide range of research techniques, including flow cytometry, microscopy, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Ab40801, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
B2883, by Merck Group (2 mentions)
Eclipse ti epifluorescence microscope, by Zeiss (2 mentions)
Gapdh, by Merck Group (1 mentions)
Mab1574, by Merck Group (1 mentions)
Irdye 680 800, by LI COR (1 mentions)
Hrp conjugated secondaries, by Cell Signaling Technology (1 mentions)
Mab5356, by Merck Group (1 mentions)
Goat anti brn3a, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab5541, by Merck Group (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Darpp32, by Abcam (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Expand high fidelity taq polymerase, by Roche (1 mentions)
Pegfp f, by Takara Bio (1 mentions)
N106 36, by Merck Group (1 mentions)
Pannav, by Merck Group (1 mentions)
Dylight 405, by Jackson ImmunoResearch (1 mentions)
14 protocols using alexa fluor 488 555 647
1
Protein Immunodetection in Neural Tissues
2
Immunohistochemistry of Retinal Cell Types
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The primary antibodies/cellular markers used are listed as follows: goat anti-Brn3a (Santa Cruz Biotechnology, 31984, 1:1000), mouse anti-calbindin (Swant, 300, 1:1000), mouse anti-CRALBP (Abcam, ab15051, 1:1000), mouse anti–HPC-1 (Sigma-Aldrich, S0664, 1:500), mouse anti-rhodopsin (Chemicon, MAB5356, 1:500), rhodamine-conjugated peanut agglutinin (PNA; Vector Laboratories, RL-1072-5, 1:500), rabbit anti-melanopsin [Advanced Targeting Systems, AB-N39 (UF008), 1:10,000], rabbit anti-melanopsin (Thermo Fisher Scientific, PA1-780, 1:500), mouse anti–SMI-32 (Covance, SMI32R, 1:1000), and sheep anti-Chx10 (Abcam, ab16141, 1:1000). Alexa Fluor 488/555/647–conjugated fluorescent secondary antibodies raised in donkey were obtained from Thermo Fisher Scientific (USA) or Jackson ImmunoResearch (USA) and used with a final dilution at 1:200.
3
Immunodetection of Neuronal Markers
4
Generation and Characterization of Membrane-Tethered Ankyrin Binding Domain
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A rabbit anti-ßIV spectrin antibody (Eurogentec) was developed against amino acid residues 15–38 of the human sequence (XP 006723369). Mouse monoclonal antibodies to ankG (1:400, N106/36 NeuroMab), Nfasc186 (1:200, L11A/41 NeuroMab) and sodium channels (pan Nav; 1:100; Sigma-Aldrich), rabbit polyclonal antibodies to GFP (1:1000; A11122 ThermoFisher), and chicken polyclonal antibodies to map2 (1:10,000; Abcam) were used. Secondary goat antibodies conjugated to Alexa Fluor 488, 555, 647 (ThermoFisher) or DyLight 405 (Jackson ImmunoResearch Laboratories) were used at 1:400 dilutions.
The nucleotide sequences of rat Nav1.2 fragment coding from amino acid 1081–1203 (wild type [WT], mutated for E1111, mutated for S1112/1123/1124/1126 or mutated for both E and the four S) were obtained by PCR amplification (Expand High Fidelity Taq polymerase, Roche Molecular Biochemicals) on pKv2.1-Nav1.2 plasmids (Bréchet et al., 2008 (link)) and inserted into the pEGFP-F (Clontech) in the 5′ of EGFP sequence. The resulting sequence encodes a chimeric protein we called membranous Ankyrin Binding Domain (mABD; see Figures6A,B ). All constructs were verified by DNA sequencing (Beckman Coulter Genomics).
The nucleotide sequences of rat Nav1.2 fragment coding from amino acid 1081–1203 (wild type [WT], mutated for E1111, mutated for S1112/1123/1124/1126 or mutated for both E and the four S) were obtained by PCR amplification (Expand High Fidelity Taq polymerase, Roche Molecular Biochemicals) on pKv2.1-Nav1.2 plasmids (Bréchet et al., 2008 (link)) and inserted into the pEGFP-F (Clontech) in the 5′ of EGFP sequence. The resulting sequence encodes a chimeric protein we called membranous Ankyrin Binding Domain (mABD; see Figures
5
Multiparametric Analysis of Cellular Organelles
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The following commercially available Abs were used: anti–acetyl-lysine (Cell Signaling Technology, catalog 9441s), anti-SAT1 (Cell Signaling Technology, catalog 61586s), anti-ATP5α (Abcam, catalog Ab14748), anti-Drosophila LAMP1 (Abcam, catalog ab30687), anti–cathepsin L (R&D Systems, catalog MAB 22591), anti-Ref(2)p (Abcam, catalog ab178440), anti–cathepsin D (Cell Signaling Technology, catalog2284), anti-p62/SQSTM1 (Novus Biologicals, catalog NBP1-48320), and secondary Abs conjugated to Alexa Fluor 488/555/647 (Thermo Fisher Scientific, catalog A11001/A21422/A21235), or Cy3/Cy5 (Rockland, catalog 611-110-122/611-104-122), or near-infrared dye 700/800 (Rockland, catalog 611-144-102/610-145-002). The following chemicals were used in the study: sodium phenylbutyrate (MilliporeSigma, catalog SML0309), glycerol phenylbutyrate (Molport, catalog cs-0017499), MG132 (MilliporeSigma, catalog M8699), N1,N11-diethylnorspermine (Santa Cruz Biotechnology, catalog sc-204114), DHE (Thermo Fisher Scientific, catalog D11347), and phalloidin conjugated with Alexa Fluor 546 (Invitrogen, catalog A22283).
6
Triple Immunofluorescent Staining of CK19, CD31, and GFP
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For triple staining of CK19, CD31, and GFP, paraffin sections were deparaffinized and pressure cooked in pH = 9 Tris-EDTA buffer for 20min. Slides were permeabilized with 0.3% Triton X-100 in PBS for 20 min at room temperature and then blocked with 5% normal donkey serum in 0.3% Triton X-100 in PBS (antibody diluent) for 30 min at room temperature. Antibodies were diluted as follows: CK19 (DSHB, TROMA-III, 1:10), CD31 (R&D Systems, AF3628, 1:100), GFP (Cell signaling, 2956, 1:100), in antibody diluent and incubated at 4°C overnight. Samples were washed three times in 0.1% Triton X-100 in PBS (wash solution) and incubated with the proper fluorescent secondary antibody (Alexa Fluor 488/555/647, Invitrogen) diluted 1:300 in antibody diluent for 2 h at room temperature. Samples were washed three times and incubated with DAPI (Sigma, B2883) for 1 min. Samples were washed three times and mounted with gelvator. Images were taken on a Nikon Eclipse Ti epifluorescence microscope or a Zeiss LSM700 confocal microscope and were analyzed with ImageJ.
7
Immunostaining of Drosophila Embryos
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Immunostainings were performed on embryos fixed in 4% formaldehyde for 20 minutes, except for E-Cadh stainings, for which embryos were fixed for 10 minutes. The following primary antibodies were used: mouse anti-EGFR Extracellular domain (1:500) from Sigma; mouse anti-Crb (Cq4) (1:20), rat anti-E-Cadh (DCAD2) 1:100 and mouse anti-Wash P3 (1:50) from Developmental Studies Hybridoma Bank, DSHB; rabbit anti-Serp (1:300) generously provided by S. Luschnig; rabbit anti-Rab11 (1:2000) and guinea pig anti-Rab5 (1:1000) were generously provided by T. Tanaka; guinea-pig anti-Uif (1:500) generously provided by R. Ward; rabbit or goat anti-GFP (1:600) from Molecular Probes and Roche used to visualise both GFP and YFP-tagged proteins; chicken anti-ßgal (1:300) from Abcam. Alexa Fluor 488, 555, 647 (Invitrogen) or Cy2-, Cy3-, Cy5-Conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1:300 in PBT 0.5% BSA. CBP (Chitin Binding Protein) was visualised as a secondary antibody at 1:300. Rhodamine-labelled 10 KDa dextran injections were used for an in vivo endocytosis assay and were performed as described in [4 (link)].
8
Multimarker Immunostaining of Mouse Tissues
9
Immunofluorescence Analysis of Cochlear Structures
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Isolated cochleae were immersed in 4% paraformaldehyde and then decalcified with 0.5 M EDTA (Solarbio, E1170). For cryosectioning, cochleae were immersed in increasing concentrations of 10–30% (w/v) sucrose (Biosharp, Amresco 0335) and then with serial mixtures of OCT (Sakura, Tissue-Tek 4583) and sucrose. The sections and whole mounts were blocked with phosphate-buffered saline blocking solution containing 5% donkey serum, 1% bovine serum albumin, 0.02% sodium azide (Sigma-Aldrich, S8032), and 0.5% Triton; incubated with diluted primary antibodies; and further with fluorescence-conjugated secondary antibodies (Alexa Fluor 488/555/647, Invitrogen). The primary antibodies were Atg7 rabbit polyclonal antibody (Thermo Fisher, PA5-35203, 1:300); Myosin VIIa mouse polyclonal antibody (Thermo Fisher, PA1-936, 1:500); Prestin goat polyclonal antibody (Santa Cruz, sc-22694, 1:200); CtBP2 mouse monoclonal antibody (BD Biosciences, 612044, 1:200); and P62 Guinea Pig polyclonal antibody (Progen, GP62-C, 1:200). The anti-fade Fluoromount-G mounting medium (SouthernBiotech, 0100-01) was used for mounting. The fluorescence images were obtained by a Zeiss LSM 710 confocal microscope. For hematoxylin staining, the whole mounts were stained with diluted hematoxylin (Solarbio, G1080) for 5 min.
10
Localization of GAA in Transplanted Mice
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For localization of GAA in the brain, transplanted mice were overnight fasted and sacrificed by transcardial perfusion with saline followed by 4% formaldehyde (FA) in phosphate-buffered saline (PBS) after administration of a ketamine/Sedator mix, 3.5 months after transplantation (n = 3). The entire brain was dissected out and overnight fixed in 4% FA before being frozen in OCT Tissue Tek (Sakura Finetek Europe). Sections were permeabilized in acetone and methanol (1:4 [v/v]) prior to blocking in 10% fetal calf serum in PBS at room temperature, and subsequently stained for GAA (anti-GAA 1:100), anti-glial fibrillary acidic protein (GFAP; Sigma-Aldrich; 1:500), and F4/80 (AbD Serotec; 1:200) overnight at 4°C. The following secondary antibodies were used: Alexa Fluor 488/555/647 (Invitrogen; 1:500). Hoechst (Sigma-Aldrich; 1:15,000) was used for nuclear staining. Tissue sections were examined by fluorescence microscopy (Leica DMRXA) connected to a Leica DFC 350FX camera.
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