Primary antibodies are listed in Table 1 . Secondary antibodies: anti-rabbit Alexa 488, anti-mouse Alexa 488, anti-rabbit Alexa 555, anti-mouse Alexa 555 from Life Technologies, (Carlsband, CA, USA), (1:500; A11008, A110296, A21428 and A21422) and anti-guinea pig Alexa 594 from Jackson ImmunoResearch (Cambridgeshire, UK), (1:500; 103-605-155).
Anti mouse alexa555
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse Alexa555 is a fluorescent labeling reagent that binds specifically to mouse immunoglobulins. It is designed for use in various immunodetection techniques, such as immunofluorescence microscopy, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
A11008, by Jackson ImmunoResearch (1 mentions)
Lsm 700, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Pgex 6p 1, by GE Healthcare (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Anti egfp, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Zeiss (1 mentions)
Volocity, by Quorum Technologies (1 mentions)
Anti cd11b, by BD (1 mentions)
Inverted microscopy, by Nikon (1 mentions)
Catalyzed signal amplification system, by Agilent Technologies (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Rabbit anti beclin1, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
16 protocols using anti mouse alexa555
1
Immunofluorescence Staining Protocol
2
Immunofluorescence Staining of KLP-7 in Embryos
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After freeze-cracking, embryos were fixed in −20°C methanol for 15 min and then incubated with 1:1,000 rabbit anti–KLP-7, a gift from K. Oegema at the University of California, San Diego, La Jolla, CA (Oegema et al., 2001 (link)), and 1:50,000 mouse anti–α-tubulin (DM1A; Sigma-Aldrich) antibodies at 4°C overnight. As previously described, the KLP-7 antibody was generated by amplifying a fragment of KLP-7 from cDNA using the primers 5′-CGCGCGAGATCTCAGAGAAAACGAGCCGAGAA-3′ and 5′-GCGCGCGAATTCTCAAGGAGCCATACGAACAGGAAC-3′. Fragments were digested with BglII-–EcoRI and cloned into pGEX6P-1 (GE Healthcare) to produce GST-KLP-7 (Oegema et al., 2001 (link)). After washing, the slides were incubated with 1:1,000 anti-rabbit–Alexa 488 and 1:1,000 anti-mouse–Alexa 555 (Life Technologies) for 2 h at room temperature. Slides were then incubated with 100 ng/ml DAPI (Roche) for 15 min before mounting. Images were collected with a confocal microscope (LSM 700; Carl Zeiss), and a z projection is shown in Fig. 1 D . A predicted but unverified in-frame splice product of klp-7(tm2143) would remove amino acids 97–364 (Fig. 1 and see the last paragraph of the preceding subsection); the peptide used as the antigen for generating the KLP-7 antisera included amino acids 14–190, and thus, the antisera might not recognize an in-frame deletion allele protein product (Oegema et al., 2001 (link)).
3
Fluorescence Microscopy of Cell Markers
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For fluorescence microscopy, cells were spotted on slides and fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, Burlington, MA) at 4ºC for 10 min, treated with 5% normal donkey serum (Jackson, USA) in PBS for 1 h, and washed with PBS. Cells were incubated with anti-EGFP (1:500, ThermoFisher Scientific), anti-CD11b (1:100, BD Pharmingen, San Diego, CA), and anti-p24 (1:50, H12 clone, NIAID U.S.A.). For secondary antibody binding, anti-chicken Alexa 488 (1:200; ThermoFisher Scientific) and anti-mouse Alexa 555 (1:100; ThermoFisher Scientific) were suspended in PBS and incubated for 1 h and washed with PBS three times then were conjugated, DAPI (ThermoFisher Scientific) staining was used as a nuclear marker and observed with fluorescence microscopy (Zeiss, Inwood, NY). Cells positive for specific markers were counted under fluorescence microscope for statistical analysis (Volocity, Quorum Technologies, Inc.). Briefly, 5 × 103 DAPI positive cells were counted for EGFP expression and presented as percent positive. For p24 staining Catalyzed Signal Amplification System (Dako, Santa Clara, CA) was used with H12 Ab clone 183-H12-5c, then observed with inverted microscopy (Nikon, Garden City, NY). Experiments were repeated at least three times.
4
Colocalization Analysis of Pacer and Beclin1
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NSC34 cells were seeded on coverslips and grown overnight. Briefly, cells were transfected and after 24 h cells washed in PBS, fixed with 4% paraformaldehyde (Merck) for 10 min at room temperature, permeabilized with 0.1% Triton X-100 (Merck, 9036-19-5) and 5% gelatin from cold water fish (Sigma, 9000-70-8) for 2 h at room temperature in a moisture chamber. Antibodies and concentrations employed were: mouse Pacer (custom-made from Abmart), 1:100; rabbit anti-Beclin1 (Santa Cruz Biotechnology, sc-11,427), 1:100. Secondary antibodies were used as follows: anti-mouse Alexa 555 (Thermo Fisher Scientific, A28180), 1:1000 and anti-rabbit Alexa 488 (Thermo Fisher Scientific, A27034), 1:1000. Nuclei were stained with Hoechst 33342 (Life Technologies, H3570)1:1000. Coverslips were mounted with Fluoromount G. Fixed cells were imaged with a Leica TCS SP8 confocal microscope. ImageJ and LAS X were employed to process the stacked images. The co-localization analysis was performed as in [42 (link)]. Briefly, confocal images were processed using CDA software. Applying the same thresholds for all experimental conditions, the software provides a coefficient of colocalization (Pearson’s correlation coefficient), which was used comparatively.
5
Immunostaining Protocol for Vimentin
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Samples were fixed with 4% PFA for 15 min at room temperature, blocked with 1% (w/v) bovine serum albumin (BSA) in PBS, and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 1 h at room temperature. Samples were incubated with primary antibody (diluted in 1% (w/v) BSA in PBS), mouse anti-vimentin (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), overnight at 4 °C. Samples were then washed three times with PBS and incubated with the following secondary antibodies: antirabbit Alexa 555 (1:200; diluted in 1% BSA in PBS), anti-mouse Alexa 555 (1:200; Thermo Fisher Scientific, Waltham, MA, USA), or Alexa Fluor 488 phalloidin (1:200; Thermo Fisher Scientific) for 2 h at room temperature. Nuclei were stained with Hoechst 33342 (2 mg/mL; Thermo Fisher Scientific) for 10 min at room temperature. Fluorescence images were acquired using a confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany) at the Soonchunhyang Biomedical Research Core Facility of KBSI.
6
Immunofluorescence Analysis of Islet Cells
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Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After antibody blocking, primary antibodies were incubated overnight at 4 °C. We used rabbit anti-insulin and mouse anti-glucagon (1:1000; Abcam, Cambridge, UK), rabbit anti-PDX1, goat anti-PDX1, mouse anti-NEUROD1, rabbit anti-MAFA (1:200; Abcam), and mouse anti-albumin (1:100, Santa Cruz Biotechnology). For secondary fluorescence labeling, the cells were incubated with anti-rabbit IgG Alexa Fluor 488, anti-rabbit IgG Alex Fluor 555, anti-goat IgG Alex Fluor 488, anti-mouse Alexa 555, and anti-mouse Alexa 488 (1:200; Thermo Fisher Scientific). ProLong Gold antifade reagent containing DAPI (Thermo Fisher Scientific) was used to stain the nuclei and for mounting. The slides were visualized using the EVOS® FL auto cell imaging system (Thermo Fisher Scientific). Under the microscope, the cells with red fluorescence were insulin or glucagon positive and blue fluorescence highlighted the nucleus and counted in 10 randomly selected fields per IPC sheet or IPC cells with total of 3 donors in each group. The results were presented as insulin- or glucagon-positive cells per 100 cells.
7
Ciona Genomic DNA and cDNA Amplification
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DNA fragments were amplified by PCR with exTaq-HS (Takara Bio) and Phusion HF (New England Biolabs) DNA polymerases from Ciona genomic DNA or cDNA. The primers that we used were summarized in Appendix Table S1 . The amplicons were subcloned into TOPO vectors (life technologies). DIG or fluorescein-labeled RNA probes were synthesized by T7 and sp6 RNA polymerases (Roche) from template DNA plasmid digested by NotI or SpeI (New England Biolabs), and were cleaned by RNeasy mini kit (QIAGEN). We followed the protocol for in situ hybridization described before (Christiaen et al, 2009b (link); Ohta and Satou, 2013 (link)). We detected fluorescein and DIG probes using TSA plus (Perkin Elmer), green (FP1168), and red (FP1170), respectively. Primer sequences are provided in Appendix Table S1 .
We used an antibody for phospho histone 3 (pH3) that was previously reported (Shirae-Kurabayashi et al, 2006 (link)) (pH3-ser10-6g3-mouse-mAb, Cell Signaling; #9706; 1:500 diluted). The primary antibody was added together with anti-DIG antibody during FISH process, and secondary antibody (anti-mouse-Alexa-555, Thermo Scientific, A-21127) was used to detect the anti-pH3 antibody after detection of ISH probe by using TSA plus (Perkin Elmer) green (FP1168).
We used an antibody for phospho histone 3 (pH3) that was previously reported (Shirae-Kurabayashi et al, 2006 (link)) (pH3-ser10-6g3-mouse-mAb, Cell Signaling; #9706; 1:500 diluted). The primary antibody was added together with anti-DIG antibody during FISH process, and secondary antibody (anti-mouse-Alexa-555, Thermo Scientific, A-21127) was used to detect the anti-pH3 antibody after detection of ISH probe by using TSA plus (Perkin Elmer) green (FP1168).
8
Whole Mount Preparation of Adult Fly Brain
9
Immunofluorescence Analysis of Muscle Tissue
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Cells and tissues were fixed in PFA 2% and processed for histology and immunocytochemistry as previously described (Gargioli et al, 2008 (link)). The primary antibodies used were mouse anti-Pax7 (DSHB) at 1:20, mouse MF20 (DSHB) at 1:2, rabbit anti-laminin (SIGMA #9393) at 1:100, rabbit anti-LacZ (Cappel) at 1:100, rat anti-VE-cadherin (clone BV13 homemade) at 1:100, mouse anti-SMA (Sigma) at 1:100, mouse anti-dystrophin (Vector) at 1:100, mouse anti-neuronal class III β-tubulin (COVANCE) and alpha-bungarotoxin Alexa594 (Molecular probes) at 50 mg/ml. The secondary antibodies used at 1:100 were anti-mouse Alexa555 (Molecular Probes), anti-rabbit Alexa488 (Molecular Probes) and anti-rat Alexa568 (Molecular Probes), Cy2-anti-mouse (Jackson), AMCA-anti-mouse (Jackson), goat anti-mouse horseradish peroxidase (HRP)-conjugated IgG (Bio-Rad) for immunohistochemistry against MF20, developing the peroxidase reaction by AEC (3-amino-9-ethylcarbazole) substrate (SIGMA). The sections were photographed with Nikon ECLIPSE 2000-TE microscope or with Olympus FV 1000 confocal laser scanning microscope for the confocal images. VE-cad-positive capillary endothelial cells were counted under fluorescence microscopy (×200) in five randomly selected fields of different sections from each sample and related to the number of muscle fibres in the same section (Díaz-Manera et al, 2010 (link)).
10
Immunofluorescence Staining of C2C12 Myoblasts
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C2C12 cells were fixed in 2% PFA over night at 4 °C, and then were permeabilized with PBS plus Triton™ X-100 (Sigma, Darmstadt, Germany) 0.3% w/v (permeabilization solution) for one hour at RT and then incubated with a blocking solution composed of PBS plus Triton™ X-100 0.1% w/v, glycine 1% w/v and goat serum NGS (Natural goat serum) 10% w/v for one hour at RT. Subsequently, the sections were incubated with a primary antibody appropriately diluted in blocking solution for one hour at RT. Two washes were performed, respectively at 15 and 5 min each, with PBS plus Triton™ X-100 0.2% w/v BSA (Bovine Serum Albumin) 1% w/v (washing solution), and then the sections were incubated for one hour at RT with a secondary antibody conjugated with a fluorophore appropriately diluted in blocking solution. After a 10 min rinse with the washing solution, DAPI was added (4′,6-diamidino-2-phenylindole), 0.1 mg/mL, in PBS for 10 min at RT. Finally, the sections were mounted with Aqua Poly/Mount (Polysciences, Warrington, PA, USA).
Primary antibody used:
Primary antibody used:
Mouse anti-MyHC (produced in our laboratory by hybridoma cells) diluted 1:2.
Anti-mouse Alexa555 (Molecular Probes) diluted 1:100.
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