Tyramide alexa fluor 594

Manufactured by Thermo Fisher Scientific

Tyramide-Alexa Fluor 594 is a fluorescent labeling reagent that can be used for signal amplification in immunohistochemistry and in situ hybridization techniques. The product contains Alexa Fluor 594 conjugated to a tyramide molecule, which can be enzymatically deposited at the site of a target antigen or nucleic acid sequence.

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Lab products found in correlation

Biotinylated goat anti rabbit igg, by Vector Laboratories (2 mentions) Lsm 880 confocal microscope, by Zeiss (2 mentions) Mowiol mounting medium, by Merck Group (1 mentions) Discovery xt processor, by Roche (1 mentions) Ezprep buffer, by Roche (1 mentions) Cc1 buffer, by Roche (1 mentions) Bond polymer refine detection kit, by Leica (1 mentions) Anti e cadherin clone 24e10, by Cell Signaling Technology (1 mentions) Anti ki 67 clone 30 9, by Roche (1 mentions) Chicken polyclonal anti gfp, by Abcam (1 mentions) 1.4 na oil immersion objective, by Zeiss (1 mentions) Airyscan, by Zeiss (1 mentions)

Close spelling variants of this product

Alexa Fluor 594 Alexa 594 Alexa Fluors

4 protocols using tyramide alexa fluor 594

Immunohistochemical Detection of HIF1-alpha

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The tissue sections were blocked for 30 minutes in 10% normal goat serum. 2% BSA in PBS. The primary antibody incubation (rabbit Hif1 alpha antibody (Chemicon, cat.#AB3883) was used in 10 μg/ml concentrations. The incubation with the primary antibody was done for 5 hours, followed by 60 minutes incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat#:PK6101) in 7.5 μg/mL dilution. The detection was performed with Blocker D, Streptavidin-HRP D (Ventana Medical Systems), followed by incubation with Tyramide-Alexa Fluor 594 (Invitrogen, cat. #T20935).

Theodoraki M.A., Rezende CO J.r., Chantarasriwong O., Corben A.D., Theodorakis E.A, & Alpaugh M.L. (2015). Spontaneously-forming spheroids as an in vitro cancer cell model for anticancer drug screening. Oncotarget, 6(25), 21255-21267.

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Immunofluorescent Detection of PARP1 in Glioblastoma

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PARP1 antigen detection in glioblastoma xenografts and mouse brain was performed at MSKCC’s Molecular Cytology Core Facility using the Discovery XT processor (Ventana Medical Systems, Tucson, AZ) and detected using immunofluorescence (IF) staining. Paraffin-embedded formalin-fixed 3 μm sections were deparaffinized with EZPrep buffer, antigen retrieval was performed with CC1 buffer (both Ventana Medical Systems), and sections were blocked for 30 min with Background Buster solution (Innovex, Richmond, CA). Anti-PARP1 rabbit polyclonal antibody (sc-7150, 0.2 μg/mL; Santa Cruz Biotechnology, Santa, Cruz, CA) was incubated for 5 h, followed by 1 h incubation with biotinylated goat anti-rabbit IgG (Vector labs, PK6106) at a 1:200 dilution. Detection was performed with Streptavidin-HRP D (from DABMap Kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 594 (T20935; Invitrogen, Carlsbad, CA) prepared according to the manufacturer’s instructions. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and coverslipped with Mowiol® mounting medium (Sigma-Aldrich, St. Louis, MO). H&E staining was performed on adjacent sections for morphological evaluation of tissue characteristics.

Salinas B., Irwin C.P., Kossatz S., Bolaender A., Chiosis G., Pillarsetty N., Weber W.A, & Reiner T. (2015). Radioiodinated PARP1 tracers for glioblastoma imaging. EJNMMI Research, 5, 46.

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Multimodal Evaluation of Tissue Markers

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Human tissues were fixed in formalin and mouse tissues were fixed in 4% paraformaldehyde for 24 h before paraffin embedding. Immunostaining was performed on 5-μm sections via standardized automated protocols on a Ventana Discovery XT machine with the following antibodies: anti-L1CAM (clone 14.10, BioLegend), anti-Ki67 (clone 30–9, Roche) and anti-E-cadherin (clone 24E10, Cell Signaling). For dual LGR5 FISH and L1CAM immunofluorescence, freshly cut 3-μm paraffin sections were stained with the RNAscope 2.5 LS Brown kit (ACD, 322100) for FISH and the Bond Polymer Refine Detection kit (Leica, DS9800) for immunofluorescence on a Leica Bond RX instrument following routine manufacturer protocol ACD 2.5 DAB and Protocol F, using RNAscope 2.5 LS probe for human LGR5 (ACD, 311028) and anti-L1CAM antibody (clone 14.10, BioLegend) with Tyramide Alexa Fluor 594 (Life Technologies, B40957) instead of the DAB step. An Ultra-High-Def mouse-on-mouse kit (StatLab) was used for mouse L1CAM staining. For Ki67 and L1CAM scoring, stained serial sections with overlapping morphology were aligned with the SIFT algorithm and the extent of immunostaining was scored with ImageJ software. L1CAM staining in Extended Data Fig. 4l,m was scored with ImageJ software. All other immunostaining was visually scored in a blinded fashion.

Ganesh K., Basnet H., Kaygusuz Y., Laughney A.M., He L., Sharma R., O’Rourke K.P., Reuter V.P., Huang Y.H., Turkekul M., Emrah E E.r., Masilionis I., Manova-Todorova K., Weiser M.R., Saltz L.B., Garcia-Aguilar J., Koche R., Lowe S.W., Pe’er D., Shia J, & Massagué J. (2020). L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer. Nature cancer, 1(1), 28-45.

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Visualizing CAR T Cell Interactions with Target Cells

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CAR T cells expressing a CAR–GFP fusion and NALM6 cells were seeded at 1:1 ratio onto poly-L-lysine-coated glass surface chamber slides. Cells were then co-cultured at 37 °C for 1 h. Cells were fixed by adding of 4% paraformaldehyde into the culture medium (final concentration 1%) and incubating for 15 min. After fixation cells were washed twice with PBS. Cells were stained using automated system Leica Bond RX Protocol F. Monoclonal mouse anti-CD19 (clone BT51E, Leica Microsystems), chicken polyclonal anti-GFP (Abcam) and rabbit polyclonal anti-CD3 (DAKO, Tyramide Alexa-Fluor-488 (Life Technologies), with Tyramide Alexa-Fluor-594 (Life Technologies), and AffiniPure Fab fragment rabbit anti-goat IgG-Alexa-Fluor-647 (Jackson Immunoreserach) were used. For nucleus staining, cells were incubated in 5 μg ml−1 DAPI/PBS solution for 5 min. Slides were mounted using Mowiol fluorescence mounting medium (Mowiol 4-88 Reagent-Calbiochem) prepared in glycerol and Tris-HCl buffer according to the manufacturer’s protocol. Cells were kept in the dark at −20 °C. For imaging, confocal z-stacks were taken at optimal imaging parameters with a LSM 880 confocal microscope with Airyscan with a 63× 1.4 NA oil immersion objective (Carl Zeiss Microimaging). ImageJ software was used to generate the figures.

Hamieh M., Dobrin A., Cabriolu A., van der Stegen S.J., Giavridis T., Mansilla-Soto J., Eyquem J., Zhao Z., Whitlock B.M., Miele M.M., Li Z., Cunanan K.M., Huse M., Hendrickson R.C., Wang X., Rivière I, & Sadelain M. (2019). CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature, 568(7750), 112-116.

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