Immunofluorescence was performed as previously described13 (link),14 (link). In brief, mouse tumor sections were de-paraffinized and rehydrated, then subjected to antigen retrieval in Target Retrieve Solution (DAKO; S1699) at 95°C for 20 min. Sections were blocked with 5% horse serum for 1 h at room temperature, incubated with anti-CD31 (1:100; Dianova; DIA-310; for mouse tissues), anti-FSP-1 (1:100; Millipore; 07–2274), anti-tdTomato (1:100; Rockland; 600-401-379) or anti-VCAM-1 (1:100; Cell Signaling Technology; 12367) antibody overnight at 4°C. For cell culture staining, the cells were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 1% Triton X-100 for 5 min. Cells were blocked with 5% horse serum for 1 h, then incubated with Alexa Fluor 488-conjugated phalloidin (1:100; Invitrogen; A12379) for 20 min. Images were acquired using an Axio Imager microscope (Zeiss) equipped with an AxioCam 506 monochrome CCD camera (Zeiss).
Anti tdtomato
Manufactured by Rockland Immunochemicals
Anti-tdTomato is a fluorescent protein detection reagent used to identify and visualize tdTomato-tagged proteins. It provides a reliable and specific method for detecting and locating tdTomato-labeled proteins in biological samples.
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Lab products found in correlation
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti fsp 1, by Merck Group (1 mentions)
Anti vcam 1, by Cell Signaling Technology (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Target retrieve solution, by Agilent Technologies (1 mentions)
Anti cd31, by Dianova (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Histomouse max kit, by Thermo Fisher Scientific (1 mentions)
Mouse anti β catenin antibody, by BD (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
2 protocols using anti tdtomato
1
Immunofluorescence Staining of Mouse Tumor Sections
2
Tissue Fixation and Immunohistochemistry
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All excised tumors were either embedded in FSC 22 (Surgipath) and flash frozen or placed in formalin, processed and embedded in paraffin. Blocks were serially sectioned and arrayed as two 5μm sections per slide. Every tenth slide was stained with hematoxylin and eosin. For more complete analysis, slides of interest were stained by immunohistochemistry for CTNNB1 (1:200 Purified Mouse Anti-β-catenin Antibody, BD Transduction Laboratories) using the HistoMouse Max Kit (Invitrogen) or by immunofluorescence for green fluorescent protein (EGFP) or red fluorescent protein (tdTomato) (primary– 1:1000 Living Colors EGFP Monoclonal Antibody, Clontech, and secondary– 1:1000 goat anti-mouse Alexa Fluor 488, Invitrogen; and/or primary– 1:200 anti-tdTomato, Rockland and secondary– 1:1000 goat anti-rabbit Alexa Fluor 568, Invitrogen) and 4',6-diamidino-2-phenylindole (DAPI) by applying ProLong Gold with DAPI mounting media (Molecular Probes). Note that staining for EGFP and tdTomato was necessary because fixation quenches the fluorescent signal.
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