Cells were labeled with 50 μM PKH26GL (Sigma-Aldrich) according to manufacturer's instructions. For in vitro assays, 3×104 labeled HCCLM3 cells were seeded into 24-well plates and immunostaining for OPN was performed after 144 hrs in culture. For in vivo assays, 5×105 labeled HCCLM3 cells were subcutaneously injected into nude mice. Six weeks later, the tumors were minced and digested with type IV collagenase (Sigma-Aldrich). Single-cell suspensions were obtained by filtration through a 70 μm filter (BD Biosciences) and immunostaining of OPN was performed. For BrdU-retaining assays, six weeks after intraperitoneal injections with BrdU (10 mg per kg body weight) three times a day for 2 days, the HCCLM3 cells-xenografted nude mice were sacrificed and tumors minced into sections and embedded in paraffin. BrdU-retaining cells were assayed by immunohistochemistry using anti-BrdU antibody (Invitrogen) and goat anti-OPN antibody (R&D Systems). Secondary antibodies were anti-mouse IgG Alexa Fluor 488 and anti-goat IgG Alexa Fluor 555 (Invitrogen).
Alexa fluor 555 anti goat igg
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Alexa Fluor 555 anti-goat IgG is a secondary antibody conjugate that binds to goat immunoglobulin G (IgG). It is used to detect and visualize the presence of goat IgG in various immunological applications, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Vectashield, by Thermo Fisher Scientific (3 mentions)
Pkh26gl, by Merck Group (1 mentions)
Anti brdu antibody, by Thermo Fisher Scientific (1 mentions)
70 μm filter, by BD (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Confocal lsm700, by Zeiss (1 mentions)
Prolong glass antifade mounting solution, by Thermo Fisher Scientific (1 mentions)
Goat anti gfp, by Thermo Fisher Scientific (1 mentions)
Ma5 13188, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
To pro 3, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde phosphate buffer solution, by Nacalai Tesque (1 mentions)
Vectashield mounting media with dapi, by Vector Laboratories (1 mentions)
Alexa fluor 594 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
6 protocols using alexa fluor 555 anti goat igg
1
Tracking OPN-expressing Tumor Cells
2
Immunofluorescence analysis of intestinal organoids
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Established organoids were passaged and replated in a 1:1 ENR:Matrigel solution; 30-μl domes were plated on Nunc Lab-Tek II Chamber Slides and incubated overnight in 200 μl medium at 37°C. Organoids were than treated with 5 ng/ml of IL-13 in ENR or simultaneously with Hpb-CM for 48 h. After stimulation, domes were fixed in 10% formalin for 30 min at room temperature (RT), and permeabilized with PBS-T (PBS + Triton 0.5%) for 15 min. The samples were blocked with 200 μl blocking solution (3% BSA in PBS) for 1 h at RT. Organoids were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at RT for 1 h. The nuclei were stained with DAPI (1 µg/ml). Specific antibodies include anti-mouse/rat Ki67 eFluor 660 (Invitrogen), anti-mouse CD326 (EpCAM) Alexa Fluor 488 (BioLegend), goat anti-GFP (Invitrogen), anti-goat IgG Alexa Fluor 555 (Invitrogen), rabbit anti-mouse Dclk (Abcam), rabbit anti-mouse Muc2 (Abcam), and goat anti-rabbit IgG Alex Fluor 555. All fluorescent images were taken on a Zeiss Confocal LSM700, using a 20×/0.8 M27 Plan-Apochromat objective, after mounting with Invitrogen Prolong Glass Antifade Mounting solution. Tile scanning (5 × 5 tiles) was performed as well as Z-stacking to generate images analyzed using Fiji software (Schindelin et al., 2012 (link)).
3
Immunostaining for Endothelial Markers
4
Immunofluorescence Analysis of hnRNPA1 and BG4
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After HCT116 cells were treated with control or hnRNPA1 siRNA for 48 h, they were fixed with 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, Kyoto, Japan) and treated with 0.5% (v/v) Triton X. These cells were further treated with 200 μg/mL RNase A for 1 h at 37 °C and then incubated overnight with 0.4 ng/μL of hnRNPA1 antibody or 4 ng/μL of BG4 antibody at 4 °C. After washing with PBS, the signals were visualized with Alexa Fluor 488 anti-mouse IgG or Alexa Fluor 555 anti-goat IgG (Thermo Fisher Scientific). The nuclei were stained with TO-PRO-3 (Thermo Fisher Scientific) using a coverslip with Vectashield (Thermo Fisher Scientific).
5
Immunofluorescence analysis of SNAIL and Jab1
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A549 cells were treated with 10 µM MG132 for 6 h. After fixation using 4% formaldehyde at room temperature for 15 min, the cells were incubated overnight at 4° C with goat polyclonal anti-SNAIL (1:200; Abcam, MA, USA) and rabbit polyclonal anti-Jab1 (COPS5) protein (1:200, Abcam). The cells were then stained with Alexa Fluor 555-anti-goat IgG (1:500, Thermo Fisher Scientific) and Alexa Fluor 488-anti-rabbit IgG (1:500, Thermo Fisher Scientific) at room temperature for 90 min and mounted using VECTASHIELD mounting media with DAPI (Vector Laboratories, Burlingame, CA).
6
Immunohistochemical Analysis of Colon and Caco-2 Cells
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For animal experiments, 24 h after intraperitoneally administering cyclosporine or the vehicle, paraffin-embedded sections of colon were obtained for immunohistochemical analysis. The sections were stained with an anti-SLC16A1 rabbit polyclonal antibody (TA 321555, OriGene, Rockville, MD, USA; 1:200) for 1 h, anti-acetyl-Histone H3 rabbit polyclonal antibody (06–599, Merck, Darmstadt, Germany; 1:500) for 1 h, anti-β1 Na+/K+-ATPase antibody (ab134280, Abcam, Cambridge, UK; 1:100) for 1 h, Alexa Fluor 488 anti-rabbit IgG (A21206, Thermo Fisher Scientific; 1:500) for 30 min, and Alexa Fluor 555 anti-goat IgG (A21432, Thermo Fisher Scientific; 1:500) for 30 min. Nuclear staining and mounting were then performed using 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; P36941, Thermo Fisher Scientific). Next, Caco-2 cells were stained with an anti-SLC16A1 mouse IgG1 monoclonal antibody (sc-365501, Santa Cruz Biotechnology; 1:200), anti- CD147 rabbit monoclonal antibody (abcam; 1:200). Alexa Fluor 488 anti-mouse IgG (A28175, Thermo Fisher Scientific; 1:200), Alexa Fluor 594 anti-rabbit IgG (A21207, Thermo Fisher Scientific; 1:200), and DAPI. The cells were stained at 14 days post-confluence, mounted on slides with coverslips, and observed using an IX73 (Olympus Co., Tokyo, Japan) and FV3000 (Olympus Co.).
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