For immunofluorescence staining, frozen xenograft tissue was sectioned, and the staining was performed according to a standard protocol. In short, the tissue was fixed in 4% PFA for 10 min; rabbit polyclonal Abs against human CD44 (Gene Tex, Irvine, CA, USA), CXCR4, and c-Met (Abcam) and mouse polyclonal Abs against RelA (Rockland, Gilbertsville, USA), Nanog (Cell Signaling), EpCam (kindly provided by Dr G. Moldenhauer), ALDH (Becton-Dickinson, Heidelberg, Germany), and CD133 (Millipore, Bergisch Gladbach, Germany) were used as the primary Abs. The nuclei were stained with DAPI (4,6-diamidino-2′-phenylindol, 1 μg/ml). Goat anti-rabbit Alexa Fluor 488 IgG, goat anti-rabbit Alexa Fluor 594 IgG, goat anti-mouse Alexa Fluor 594 IgG, and goat anti-mouse Alexa Fluor 488 IgG (Invitrogen, Camarillo, CA, USA) were used as the secondary Abs.
Alexa fluor 488 igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 IgG is a fluorescent dye conjugated to an IgG antibody. It is a fluorescent label used for various biological applications involving detection and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 igg, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Alexa fluor 555 igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568 igg, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 647 igg, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Anti nephrin, by R&D Systems (2 mentions)
Anti synaptopodin, by Synaptic Systems (2 mentions)
Hoechst, by Beyotime (2 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Slowfade gold reagent, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Normal donkey serum (nds), by Merck Group (2 mentions)
Slow fade antifade kit, by Olympus (2 mentions)
Fluorescence microscopy, by Olympus (2 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Cd133, by Merck Group (1 mentions)
51 protocols using alexa fluor 488 igg
1
Immunofluorescence Staining of Xenograft Tissue
2
VSMC Calcification and Microtubule Dynamics
3
Autoantibody Detection in Scurfy Mice
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Serum samples were taken from scurfy and WT mice on day 21 of life. For evaluation of autoantibodies by immunofluorescence (IF), sera were diluted (as indicated in the figure legends) and added to slides precoated with either Crithidia luciliae (dsDNA) or HEp-20-10 cells and primate liver cells, respectively (ANAs) (all from EUROIMMUN, Lübeck, Germany). As a secondary Ab, goat-anti mouse immunoglobulin G (IgG) Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA), diluted 1:500 in phosphate-buffered saline (PBS), was used. For semiquantitative analyses, the slides were scored according to fluorescence intensity as follows: 0 = no positive staining, 1 = weakly positive staining, 2 = intermediate positive staining and 3 = strongly positive staining.
Anti-histone Abs were measured by enzyme-linked immunosorbent assay (ELISA) (Inova Diagnostics, San Diego, CA, USA). The results are presented in units per milliliter. Horseradish peroxidase–conjugated goat anti-rat Abs (1:2,000 dilution; SouthernBiotech, Birmingham, AL, USA) served as secondary Abs. Further analysis was performed by immunoblotting as described elsewhere [29 (link),30 (link)].
Anti-histone Abs were measured by enzyme-linked immunosorbent assay (ELISA) (Inova Diagnostics, San Diego, CA, USA). The results are presented in units per milliliter. Horseradish peroxidase–conjugated goat anti-rat Abs (1:2,000 dilution; SouthernBiotech, Birmingham, AL, USA) served as secondary Abs. Further analysis was performed by immunoblotting as described elsewhere [29 (link),30 (link)].
4
Immunofluorescence Analysis of Stem Cell Markers
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Freshly purified KLS34− cells were sorted by FACS, and cytospins were fixed in 4% paraformaldehyde. The slides were then washed in PBS + 0.1% Tween, blocked with 20% normal goat serum, and stained with mouse anti-active β−catenin (Upstate Biotechnology) or mouse IgG isotype control (BD Pharmingen). The slides were washed in PBS-Tween (0.1%) and stained with donkey anti-mouse IgG-TRITC (Jackson ImmunoResearch) or IgG-Alexa Fluor 488 (Invitrogen). Cell images were taken with a Zeiss 410 Axiovert microscope. To evaluate nuclear γ-H2A.X and 53BP1 staining, cells were treated as described above and then stained with rabbit polyclonal antibody to phosphorylated H2A.X (Abcam) or 53BP1 and then washed and labeled with Alexa Fluor 488 secondary antibody (Invitrogen). For bone staining, fresh bone specimens from control and irradiated mice were decalcified, infiltrated with sucrose, and embedded in OCT medium (Tissue-Tek). The frozen sections were fixed in acetone, washed in PBS-T, and blocked with 20% normal donkey serum (Jackson ImmunoResearch). The tissue sections were stained with anti-Wnt10b (Santa Cruz Biotechnology) or the appropriate isotype control followed by donkey anti-goat Alexa Fluor 594 (Invitrogen). The nuclear dye DAPI (Invitrogen) was included in all stains.
5
Sciatic Nerve Immunohistochemistry Protocol
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The distal sciatic nerve samples were fixed with 4% paraformaldehyde and dehydrated in 30% sucrose solution. Sections were cut using a cryostat to a thickness of 12 µm and mounted onto slides. The sections were rinsed in PBS, permeabilized in 0.3% Triton X-100, 5% goat serum, and 1% BSA in PBS, and then stained. The sections were incubated with mouse monoclonal anti-S100 (1:400, Sigma) and Spp1 (1:50, Santa Cruz) antibodies at 4 °C for 12 h, and then incubated with goat anti-mouse or goat anti-rabbit IgG Cy3 (1:400, Sigma) and IgG Alexa Fluor 488 (1:400, Invitrogen) at room temperature for 2 h. The sections were counterstained with Hoechst 33342 for 5 min. All samples were observed under a fluorescence microscope. Images were acquired using a laser microscope (FV10i-oil, Tokyo, Japan).
6
Visualizing Anti-CSP mAb Localization in Wildtype SPZ
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Immunohistochemistry was performed to visualize the localization of anti-CSP mAbs. Wildtype SPZ were incubated with a concentration range ( 0.5, 1, 2.5, 5, 10, 25, 50 µg/ml) of the anti-CSP mAbs 2A10 or 3SP2 in RPMI + 10% FBS for 30 min at RT and subsequently 0.5*105 SPZ were added to a confocal dish without any precoating (ø14 mm; MatTek Corporation) and covered with a coverslip (ø12 mm; VWR Avantor). Subsequently, the secondary antibody (goat anti-mouse IgG Alexa Fluor 488, 1:500 in PBS; Invitrogen) was added and incubated for 45 min at RT. The SPZ were counterstained with Cy5-Methyl-Methyl (50 nm; whole body staining) and Hoechst (10 µg/ml; nucleus staining). Finally, the samples were mounted with Prolong Gold Dapi (Invitrogen) and examined using a Leica TCS (true confocal scanning) SP8X WLL (white light laser) microscope (Leica Microsystems) with a 100 × objective (HCX PL APO 100x/1.40–0.70 OIL CS). The Alexa 488 dye conjugated to the secondary antibody was excited at 488 nm (emission: 500–550 nm), Cy5-Methyl-Methyl was excited at 633 nm (emission: 650–700 nm) and Hoechst was excited at 405 nm (emission: 420–470).
7
Immunostaining of Cryopreserved Tissue Sections
8
Immunohistochemical Analysis of Drosophila Indirect Flight Muscles
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Adult virgin female flies were fixed in 4% paraformaldehyde for 48 h at 4°C. Indirect flight muscles were dissected in 1X PBS, permeabilized in 1X PBS 0.15% Triton‐X 100, and blocked in NGS +0.15% Triton‐X 100. Samples were incubated in primary antibody at 4°C overnight, washed in 1X PBS 0.15% Triton‐X 100, and incubated in secondary antibody at room temperature for 2 h. Samples were mounted in Vectashield mounting medium (Vectorlabs) and visualized using a Leica SP8 laser scanning confocal microscope. Antibodies: rabbit anti‐GFP 1:400 (Invitrogen), mouse anti‐FLAG M2 1:1000 (Sigma), rabbit anti‐stv 1:1000 (gift of Jög Höhfeld), rat anti‐α actinin 1:100 (Abcam), rabbit anti‐ubiquitin linkage‐specific K63 1:200 (Abcam), mouse anti‐Lam Dm0 1:100 (DSHB), IgG‐ Alexa Fluor 488 1:200 (Life Technologies), anti‐mouse IgG‐ Alexa Fluor 568 1:500 (Life Technologies), anti‐rabbit IgG‐ Alexa Fluor 488 1:500 (Life Technologies), syto‐24 1:10,000 (Molecular Probes) and Rhodamine Phalloidin 1:2000 (Molecular Probes).
9
Immunofluorescence Analysis of Lipid Droplets
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Cells were fixed by 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature, permeabilized with 0.2% TritonX-100 in PBS for 10 min, and blocked with 3% BSA in PBS. Slides were stained with anti-Core C7-50 (1:200 dilution) at 4 °C overnight, washed three times with PBS, and stained with IgG-Alexa Fluor-488 (1:250 dilution) (Life Technologies) at room temperature for 2 h. After washing three times with PBS, Oil Red O (Sigma, USA) staining was performed as previously described58 (link). The slides were mounted by Prolong Antifade (Life Technologies). Images were collected using a confocal microscope (Zeiss, LSM710) and processed using Adobe-Photoshop-CS5 software.
10
Immunofluorescence Staining of Selectin-Like Antigens
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All cell lines and respective KO cell models were seeded in 12-well chamber slides (ibidi) or in 13 mm coverslips (Marienfeld). The cells were washed with phosphate-buffered saline (PBS) and fixed with methanol during 20 min at -20 °C. Blocking of immunoglobulins cross-reaction was performed using goat serum (1:5) (Dako) in 10% BSA in PBS at RT for 30 min, followed by primary antibody incubation at 4 °C overnight. Slides were then incubated with fluorescently-labeled secondary antibodies anti-mouse IgM Alexa Fluor® 594 (1:500) or IgG Alexa Fluor® 488 (1:750) (Thermo Fisher Scientific) for 1 h at RT. Nuclear counterstaining was performed by incubating cells with 4`,6`-diamino-2fenil-indol (DAPI) for 10 min and slides or coverslips were mounted with VectaShield (Vector Laboratories). The fluorescent signal was examined using a fluorescence microscope and images were acquired with Zeiss Axio Imager Z1, Zeiss AxioCam MR version 3.0, and the AxioVisionRel (version 4.8) software (Carl Zeiss). Images were acquired under 200x and 400x (Fig. 5 and S3 ) magnifications.
The primary antibodies used were: SLeX (CSLEX, 1:250, BD Bioscience,), SLeA (CA19-9, 1:200, Abcam,), LeX (SH1, 1:5, [22 ]) and LeA (CA3F4 1:5, [23 ]).
The primary antibodies used were: SLeX (CSLEX, 1:250, BD Bioscience,), SLeA (CA19-9, 1:200, Abcam,), LeX (SH1, 1:5, [22 ]) and LeA (CA3F4 1:5, [23 ]).
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