Alexa fluor 488 goat anti rabbit secondary

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

Alexa Fluor 488 goat anti-rabbit secondary is a fluorophore-conjugated secondary antibody used for detection and visualization in various immunochemical techniques. It specifically binds to rabbit primary antibodies and emits green fluorescence upon excitation.

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Alexa Fluor 488 goat anti-rabbit (709 citations) Alexa Fluor 488 anti-rabbit (273 citations) Goat anti-rabbit Alexa 488 (232 citations) Alexa Fluor 488 goat anti (9 citations) 488 goat anti-rabbit (9 citations) Alexa Fluor goat anti-rabbit (8 citations) Alexa Fluor 488 anti (6 citations) Alexa Fluor anti-rabbit (4 citations) Alexa-488 anti-goat (3 citations) Alexa anti-rabbit 488 (3 citations)

8 protocols using alexa fluor 488 goat anti rabbit secondary

Metastasis Quantification via Immunofluorescence

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Staining was conducted as previously published [11 (link), 22 (link)]. RFP staining: rabbit anti-RFP (Abcam, Cambridge, UK) 1:30 dilution, Alexa Fluor® 488 goat anti-rabbit secondary (Invitrogen); CDH1 staining: rabbit E-cadherin antibody (24E10; Cell Signaling Technology, Boston, MA, USA) 1:400 dilution. Internal negative controls were exposed to rabbit IgG or 10% goat serum rather than primary antibody. Images were captured on a Nikon TE2000 inverted microscope with IPLab software (BD Biosciences, Rockville, MD, USA), original magnification at 200× (RPF) or 400× (CDH1).
Metastases were calculated as a ratio of the number of RFP-positive cells versus the total amount of cells (determined by DAPI nuclear stain) per field of view. Points represent the ratio of RPF-positive cells versus DAPI for each section examined with mean for each group represented as horizontal black bar. SEM for each group indicated in red.

Rhodes L.V., Tate C.R., Segar H.C., Burks H.E., Phamduy T.B., Hoang V., Elliott S., Gilliam D., Pounder F.N., Anbalagan M., Chrisey D.B., Rowan B.G., Burow M.E, & Collins-Burow B.M. (2014). Suppression of triple-negative breast cancer metastasis by pan-DAC inhibitor panobinostat via inhibition of ZEB family of EMT master regulators. Breast cancer research and treatment, 145(3), 593-604.

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Analyzing Tight Junction Proteins

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The following antibodies were used for protein analysis by western blot and immunofluorescence: mouse anti-β-actin (1:50,000^{21 ,22 (link)}) (Sigma, Saint Louis, MO, #A5441), mouse anti-claudin 1 (Invitrogen, Carlsbad, CA, #37-4900), mouse anti-claudin 2 (Invitrogen, Carlsbad, CA, #32-5600), rabbit anti-claudin 3 (Invitrogen, Carlsbad, CA, #341700), mouse anti-claudin 4 (Invitrogen, Carlsbad, CA, #329400). The following secondary antibodies were used for detection by immunofluorescence microscopy: Alexa Fluor 594 goat anti-mouse (Invitrogen, Carlsbad, CA, #A-11032) and Alexa Fluor 488 goat anti-rabbit secondary (Invitrogen, Carlsbad, CA, #A-11034).

Ares G., Buonpane C., Sincavage J., Yuan C., Wood D.R, & Hunter C.J. (2019). Caveolin 1 is Associated with Upregulated Claudin 2 in Necrotizing Enterocolitis. Scientific Reports, 9, 4982.

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Immunofluorescence Imaging of Cell Adhesion Proteins

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The following antibodies were used for immunofluorescence (IF): anti-E-cadherin (1:100; Cell Signaling), anti-β-catenin (1:100; Sigma-Aldrich), anti-Lamp1 (1:100; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; Life Technologies). IF was performed as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek) and were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was performed using Volocity software (PerkinElmer).

Chen R., Ram A., Albeck J.G, & Overholtzer M. (2021). Entosis is induced by ultraviolet radiation. iScience, 24(8), 102902.

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Immunofluorescence Microscopy of Cellular Targets

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Immunofluorescence was performed on cells cultured on glass‐bottom dishes (P35G‐1.5‐20‐C, MatTek, Ashland, MA, USA), as described previously.⁷ Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20℃, followed by three 5‐min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 h, followed by incubation with primary antibodies at 4℃ overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (D1306, Life Technologies, Carlsbad, CA, USA). Confocal microscopy was performed with the Ultraview Vox spinning‐disk confocal system (PerkinElmer) equipped with a Yokogawa CSU‐X1 spinning disk head and an electron‐multiplying charge‐coupled device camera (Hamamatsu C9100‐13) coupled to a Nikon Ti‐E microsope; image analysis was done using Volocity software (PerkinElmer). The following antibodies were used for immunofluorescence: anti‐phospho‐mTOR (Ser2448) (5536, Cell Signaling, Beverly, MA, USA), anti‐LAMP1 (555798, BD Biosciences, San Jose, CA, USA), anti‐BrdU (5292, Cell Signaling, Beverly, MA, USA), Alexa Fluor 568 goat anti‐mouse secondary (A‐11031, Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 488 goat anti‐rabbit secondary (A‐11034, Life Technologies, Carlsbad, CA, USA).

Kim S.E., Zhang J., Jiang E, & Overholtzer M. (2021). Amino acids and mechanistic target of rapamycin regulate the fate of live engulfed cells. The FASEB Journal, 35(10), e21909.

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Immunofluorescence Staining of Embryos

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Embryos were fixed at the appropriate stage in 4% paraformaldehyde, permeabilized in TBST (TBS+0.1% Triton X-100), and blocked in TBST+2% BSA. Anti-laminin antibody (Sigma #L9393) was diluted 1:100, anti-vinculin antibody (Sigma #V4505) was diluted 1:100, anti-activated caspase-3 antibody (BD Pharmingen #559565) was diluted 1:200, anti-aPKC (PKC ζ (C-20) Santa Cruz Biotechnology #sc-216) was diluted 1:100, anti-fibronectin antibody (Sigma #F3648) was diluted 1:100. Alexa Fluor 488 goat anti-rabbit secondary (Life Technologies, A-11008) or Alexa Fluor 488 goat anti-mouse secondary (Life Technologies, A-11001) was coincubated with 1 µM TOPRO-3 iodide (Life Technologies, T3605). Embryos were cleared in 70% glycerol for imaging.

Bryan C.D., Chien C.B, & Kwan K.M. (2016). Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis. Developmental biology, 416(2), 324-337.

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Immunofluorescence Imaging of β-Catenin and Lamp1

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The following antibodies were used for immunofluorescence (IF): anti-β-catenin (1:100; C2206; Sigma-Aldrich), anti-Lamp1 (1:100; 555798; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; A-11031; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; A-11034; Life Technologies). IF was performed on cells cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek), as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; D1306; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was done using Volocity software (PerkinElmer).

Hamann J.C., Surcel A., Chen R., Teragawa C., Albeck J.G., Robinson D.N, & Overholtzer M. (2017). Entosis Is Induced by Glucose Starvation. Cell reports, 20(1), 201-210.

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Immunofluorescence Imaging of β-Catenin and Lamp1

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Hamann J.C., Surcel A., Chen R., Teragawa C., Albeck J.G., Robinson D.N, & Overholtzer M. (2017). Entosis Is Induced by Glucose Starvation. Cell reports, 20(1), 201-210.

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Ex Vivo Tumor Analysis Protocol

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For ex vivo analysis, mice were sacrificed on the specified day and the entire tumor excised and either lysed for Western blot analysis or fixed in 10% formalin and paraffin embedded for immunohistochemical or immunofluorescence staining. For Western blot analyses, anti-CD8 (Ab108292, Abcam, Cambridge, MA), anti-CD3 (sc-20047, Santa Cruz Biotechnology, Dallas, TX, anti-CD4 (sc-19643, Santa Cruz), anti-FoxP3 (12653s, Cell Signaling Technologies, Danvers, MA), anti-pSTAT (700349, ThermoFisher, Waltham, MA), anti-Granzyme B (4275s, Cell Signaling Technologies) and anti-β-actin (Cell Signaling Technologies 4970S) were used at the manufacturer’s recommended concentration. For immunohistochemistry analysis, granzyme B (Ab4059, Abcam) and CD3 (Ab56090, Abcam) antibodies were detected using biotinylated goat-anti-rabbit antibodies with the Signal Stain Boost IHC Detection Reagent (Abcam). For immunofluorescence staining, granzyme B was detected with Ab4059 and AlexaFluor 488 goat anti-rabbit secondary (Life Technologies); CD3 was detected with anti-CD3 clone PC3/188A (Santa Cruz) and AlexaFluor 594 goat anti-mouse secondary (Life Technologies).

Larimer B.M., Wehrenberg-Klee E., Dubois F., Mehta A., Kalomeris T., Flaherty K., Boland G, & Mahmood U. (2017). Granzyme B PET Imaging as a Predictive Biomarker of Immunotherapy Response. Cancer research, 77(9), 2318-2327.

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