Anti goat alexafluor594

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Anti-goat AlexaFluor594 is a fluorescent-dye-labeled secondary antibody that binds to goat primary antibodies. It is designed for use in immunofluorescence and other fluorescence-based applications.

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7 protocols using anti goat alexafluor594

Multimodal Tumor Immunofluorescence Analysis

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EDA expression was assessed on ice-cold acetone fixed 8-µm cryostat sections of WEHI-164, CT26, F9 and LLC stained with IL2-F8-TNF^mut (final concentration 5µg/mL), as negative control IL2-KSF-TNF^mut (specific for an irrelevant antigen) was used. Both antibodies were detected with rat anti-IL2 (eBioscience 14-7022-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used.
For ex-vivo immunofluorescence analysis, mice bearing CT26 or LLC lesions were injected once with IL2-F8-TNF^mut + anti-PD-L1 or saline according to the therapy schedule and sacrificed 24h after injection. Tumors were excised and embedded in cryoembedding medium (Thermo Scientific) and cryostat sections (8µm) were stained using the following antibodies: rabbit anti-CD4 (SinoBiological 50134-R001), rabbit anti-CD8 (SinoBiological 50389-R208), rabbit anti-FoxP3 (Invitrogen 700914), rabbit anti-Natural Cytotoxicity Receptor 1 (NCR1) (Abcam ab214468), goat anti-CD31 (R&D AF3628) and detected with anti-rat AlexaFluor488 (Invitrogen A21208), anti-rabbit AlexaFluor488 (Invitrogen A11008), anti-goat AlexaFluor594 (Invitrogen A11058). Slides were mounted with fluorescent mounting medium and analysed with Axioskop2 mot plus microscope (Zeiss).

De Luca R, & Neri D. (2018). Potentiation of PD-L1 blockade with a potency-matched dual cytokine antibody-fusion protein leads to cancer eradication in BALB/c-derived tumors but not in other mouse strains. Cancer immunology, immunotherapy : CII, 67(9), 1381-1391.

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Immunofluorescent Detection of Antigen Expression

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Antigen expression was confirmed on ice-cold acetone fixed 8 μm cryostat sections of SKRC52 and CT26-CAIX stained with IL2-XE114-TNF^mut and IL2-F8-TNF^mut (final concentration 5 μg/mL) and detected with rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. IL2-KSF-TNF^mut (specific for an irrelevant antigen) was used as negative control. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).
For ex vivo immunofluorescence analysis, mice were injected with 50–60 μg IL2-XE114-TNF^mut, IL2-F8-TNF^mut, or IL2-KSF-TNF^mut and sacrificed 24 h after injection. Organs were excised and embedded in cryo-embedding medium (Thermo Scientific) and cryostat section (10 μm) were stained using the following antibodies: rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).

De Luca R., Gouyou B., Ongaro T., Villa A., Ziffels B., Sannino A., Buttinoni G., Galeazzi S., Mazzacuva M, & Neri D. (2019). A Novel Fully-Human Potency-Matched Dual Cytokine-Antibody Fusion Protein Targets Carbonic Anhydrase IX in Renal Cell Carcinomas. Frontiers in Oncology, 9, 1228.

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Immunohistochemical Analysis of Chemokines

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Adjacent 1-in-12 series of coronal free-floating sections (n = 5 per genotype and age) were incubated with 50 mM NH₄Cl for 30 min to reduce non-specific background staining and blocked with 15 % fetal calf serum (FCS) diluted in TBS/0.3 %Triton X-100 (TTX) for 1 h. The sections were incubated with the primary antibodies rabbit anti-ionized calcium-binding adaptor molecule 1 (IBA1; Wako) combined with goat anti-CXCL13, goat anti-CXCL10 (both R&D Systems), or rabbit anti-CXCL1 (Novus Biologicals) in 10 % FCS/TTX for 72 h at 4 °C. The secondary antibodies anti-rabbit Alexa Fluor 488 and anti-goat Alexa Fluor 594 (Invitrogen) were applied for 2 h at RT, and mounted sections were examined using a fluorescence microscope.

Okuneva O., Li Z., Körber I., Tegelberg S., Joensuu T., Tian L, & Lehesjoki A.E. (2016). Brain inflammation is accompanied by peripheral inflammation in Cstb−/− mice, a model for progressive myoclonus epilepsy. Journal of Neuroinflammation, 13, 298.

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Immunofluorescent Staining of Tumor Antigens

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The FAP and EDA expressions were confirmed on 8 µm cryostat sections of SKRC52-hFAP fixed in ice-cold acetone. The primary antibodies utilized were 7NP2 IgG1, F8 IgG2a, KSF IgG4 (at 5 µg/mL) (the KSF antibody is specific for an irrelevant antigen), and rat anti-mouse CD31 for the staining of blood vessels (R&D AF3628). Detection was performed with goat anti-mouse Alexa 594 (Invitrogen A11005), goat anti-human Alexa 488 (Invitrogen A11013), and donkey anti-rat Alexa 594 (Invitrogen A11058). The tumor antigen expression was confirmed utilizing IL2-7NP2-TNF^mut, IL2-F8-TNF^mut, and IL2-KSF-TNF^mut. In this case, the detection was performed with rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining, goat anti-mouse CD31 (R&D, Minneapolis, MI, USA, catalog: AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. The cell nuclei were stained with DAPI (Invitrogen; D1306). Finally, the slides were mounted with a fluorescent mounting medium (Dako, Santa Clara, CA, USA) and analyzed with a wide-field Leica TIRF microscope using Leica LAS X Life Science Microscope Software (v 1.4.4, Wetzlar, Germany).

Prodi E., Comacchio C., Gilardoni E., Di Nitto C., Puca E., Neri D, & De Luca R. (2023). An Antibody Targeting Fibroblast Activation Protein Simultaneously Fused to Interleukin-2 and Tumor Necrosis Factor Selectively Localizes to Neoplastic Lesions. Antibodies, 12(2), 29.

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Quantifying Immune Infiltration in Tumor Sections

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Infiltration of NK cells and macrophages in the B16F10 tumor masses was assessed by immunofluorescence studies on ice-cold acetone-fixed 8μm thick tumor sections. Rabbit anti-Asialo GM1 (Wako Chemicals) and rat F4/80 (BM8, eBiosciences) were detected with anti-rabbit Alexa488 (Invitrogen; A21206) and anti-rat Alexa 488 (Invitrogen; A21208), respectively. Blood vessels were stained with goat anti-CD31 (R&D Systems; AF3628) and anti-goat AlexaFluor594 (Invitrogen; A11058) antibodies. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).

Murer P., Kiefer J.D., Plüss L., Matasci M., Bluemich S, & Neri D. (2018). Targeted delivery of TNF potentiates the antibody-dependent cell-mediated cytotoxicity of an anti-melanoma immunoglobulin. The Journal of investigative dermatology, 139(6), 1339-1348.

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Quantifying Proliferating Microglia via Immunofluorescence

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To identify proliferating microglia, we performed double-labeling immunofluorescence for Iba-1 and Ki-67. Antigen retrieval and blocking were conducted as described above, followed by overnight incubation with goat polyclonal Anti-Iba1 (Abcam, Cambridge, MA, cat. #5076, dilution 1:100) and rat monoclonal anti-Ki67 (LifeSpan Biosciences, Seattle, WA, cat. #LS-C175347, dilution 1:50). The following day, sections were washed and incubated with anti-goat Alexa Fluor 594 (Life technologies, Carlsbad, CA, cat. #A-11058, dilution 1:200) and anti-rat Alexa Fluor 488 (Life technologies, Carlsbad, CA, cat. #A-21470, dilution 1:200) for 2 h at room temperature, followed by washing and incubation with DAPI (Life technologies, Carlsbad, CA, cat. #D1306, dilution 1:2000) for 15 min at room temperature. Sections were washed and mounted with ProLong Gold (Life technologies, Carlsbad, CA, cat. #P36930). Stained slides were imaged on confocal microscopy for co-localization at ×40x magnification with 1024 × 1024 pixel resolution on a Nikon Eclipse A1RSi inverted microscope (Nikon Instruments Inc, Melville, NY).

Schmidt A.F., Kannan P.S., Chougnet C.A., Danzer S.C., Miller L.A., Jobe A.H, & Kallapur S.G. (2016). Intra-amniotic LPS causes acute neuroinflammation in preterm rhesus macaques. Journal of Neuroinflammation, 13(1), 238.

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Immunofluorescent Analysis of IL-33 and F4/80 in Colon Sections

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For immunofluorescent staining of IL-33 with F4/80 on colon sections, sections were deparaffinized, rehydrated, and antigen was unmasked in a high pH buffer (Vector Labs, Burlingame, CA). Sections were stained with goat anti-mouse IL-33 (AF3626, R&D Systems, Minneapolis, MN) followed by anti-goat-Alexa Fluor-594 (Life Technologies, Carlsbad, CA) with anti-F4/80-FITC (11-4801-11, eBioscience, San Diego, CA) and sections were counterstained with Vectashield with DAPI (Vector Laboratories, Burlingame, CA).
T84 cells grown on coverslips were subsequently fixed with 4% paraformaldehyde for 30 minutes. Cells were blocked with goat serum and then stained with rabbit anti-ST2 (ab25877, AbCam, Cambridge, MA) followed by goat anti-rabbit-Alexa Fluor488 (Jackson Immunoresearch, West Grove, PA), and Alexa Fluor635-Phalloidin (Life Technologies, Carlsbad, CA) and counterstained with Vectashield with DAPI (Vector Laboratories, Burlingame, CA).

Waddell A., Vallance J.E., Moore P.D., Hummel A.T., Wu D., Shanmukhappa S.K., Fei L., Washington M.K., Minar P., Coburn L.A., Nakae S., Wilson K.T., Denson L.A., Hogan S.P, & Rosen M.J. (2015). IL-33 signaling protects from murine oxazolone colitis by supporting intestinal epithelial function. Inflammatory bowel diseases, 21(12), 2737-2746.

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