Alexa fluor 488 donkey anti sheep

Manufactured by Thermo Fisher Scientific

Alexa Fluor 488 donkey anti-sheep is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and bind to sheep primary antibodies, allowing for fluorescent visualization and analysis in various immunoassay techniques.

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6 protocols using alexa fluor 488 donkey anti sheep

Antibody Reagents for INTS3 and hSSB1 Detection

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The primary antibody against
INTS3 (antirabbit, cat no. A302-050A, Bethyl) was purchased from Thermo
Fisher Scientific, and an antigoat IgG isotype control antibody was
purchased from Sigma-Aldrich. The antigoat hSSB1 antibody was generated
in-house as described previously.^{65 (link)} The
secondary antibodies, donkey antirabbit (cat no. 926-68073) and donkey
antigoat (cat no. 926-62214), were from LI-COR, Inc. The secondary
antibodies for immunofluorescence, Alexa Fluor 488 donkey antisheep
(Invitrogen, cat no. A11015), Alexa Fluor 488 donkey antimouse (Invitrogen,
cat no. A21202), and Alexa Fluor 594 donkey antirabbit (Invitrogen,
cat no. A23753) antibodies, were purchased from Life Technologies.
4′-6-Diamidino-2-phenylindole (DAPI) was from Life Technologies.
A complete EDTA-free protease inhibitor mixture was obtained from
Roche Applied Sciences. The Pierce bicinchoninic acid (BCA) protein
assay kit (cat no. 23225) was purchased from Thermo Fisher Scientific.

Barbhuiya T.K., Beard S., Shah E.T., Mason S., Bolderson E., O’Byrne K., Guddat L.W., Richard D.J., Adams M.N, & Gandhi N.S. (2024). Targeting the hSSB1-INTS3 Interface: A Computational Screening Driven Approach to Identify Potential Modulators. ACS Omega, 9(7), 8362-8373.

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Optimized Immunofluorescence Imaging Reagents

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Pharmacological inhibitors were prepared in either DMSO or 20 % acetonitrile/water according to the manufacturer’s recommendations and used at the indicated concentrations. EIPA, CPZ, Gen, and Rot were purchased from Sigma Aldrich. The Rac1 inhibitor W56 was purchased from Tocris Bioscience.
Specific antibodies including mouse anti-beta actin (ab8226), rabbit anti-EEA1 antibody (ab2900), Anti-LAMP1 [H4A3], anti-beta actin antibody [AC-15], anti-beta tubulin antibody, anti-neuron specific beta III tubulin were purchased from Abcam. Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, streptavidin Alexa Fluor 633 conjugate, streptavidin Alexa Fluor 488 conjugate, Alexa Fluor 488 donkey anti-sheep, Alexa Fluor 488 donkey anti-rabbit, SYTOX Red dead cell stain, FM® 1-43FX fixable analogue of FM® 1–43 membrane stain were purchased from Invitrogen Life Technologies. Donkey anti-sheep/goat IgG HRP conjugate and goat anti-mouse IgM + IgG + IgA (H + L) HRP conjugates were purchased from Millipore.
Sheep anti-SOD1 was purchased from Thermo Fisher Scientific. Mouse monoclonal anti-human TARDBP antibody (clone k1B8) was purchased from Abnova. Anti-BiP/GRP78 was purchased from BD Transduction Laboratories. FITC-conjugated sheep anti-mouse was purchased from Silenus. RedDot 2 was obtained from Biotium. Goat Anti-Rabbit IgG (H + L)-HRP Conjugate was obtained from Bio-Rad.

Zeineddine R., Pundavela J.F., Corcoran L., Stewart E.M., Do-Ha D., Bax M., Guillemin G., Vine K.L., Hatters D.M., Ecroyd H., Dobson C.M., Turner B.J., Ooi L., Wilson M.R., Cashman N.R, & Yerbury J.J. (2015). SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation. Molecular Neurodegeneration, 10, 57.

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Dual-color in situ hybridization in Drosophila embryos

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Embryos were dechorionated and fixed in fixation buffer (0.5x PBS, 25 mM EGTA, 4% formaldehyde and 50% Heptane) for 20 min at room temperature. Antisense RNA probes labeled with digoxigenin (DIG RNA Labeling Mix 10 × conc, Roche) and biotin (Biotin RNA Labeling Mix 10 × conc, Roche) were used to detect lacZ and snail RNAs, respectively. Hybridization was performed at 55 °C overnight in hybridization buffer (50% formamide, 5x SSC, 50 μg/ml Heparin, 100 μg/ml salmon sperm DNA, 0.1% Tween-20). Subsequently, embryos were washed with hybridization buffer at 55 °C and incubated with Western Blocking Buffer (Roche) at room temperature for one hour. Then, embryos were incubated with sheep anti-digoxigenin (Roche) and mouse anti-biotin primary antibodies (Invitrogen) at 4 °C for overnight, followed by incubation with Alexa Fluor 488 donkey anti-sheep (Invitrogen) and Alexa Flour 555 goat anti-mouse (Invitrogen) fluorescent secondary antibodies at room temperature for two hours. DNA was stained with Hoechst 33342 (Thermo Fisher Scientific), and embryos were mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Imaging was performed on a Zeiss LSM 880 confocal microscope. Plan-Apochromat 20x / 0.8 N.A. objective was used. Images were captured in 16 bit.

Lim B., Heist T., Levine M, & Fukaya T. (2018). Visualization of Transvection in Living Drosophila Embryos. Molecular cell, 70(2), 287-296.e6.

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Antibody Characterization and Validation

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The applied primary antibodies are listed in Table 2. The three anti-albumin antibodies performed equally well in immunoblotting, with lower molecular bands appearing on prolonged exposure for the sheep anti-albumin. Double immunofluorescence labeling shows robust co-localization of the pairwise staining with the rabbit and sheep antibodies as well as with the rabbit and goat antibodies (S1 Fig). Secondary antibodies were: HRP-conjugated goat anti-rabbit or goat anti-mouse IgG (DAKO A/S Denmark), AlexaFluor-488 donkey anti-rabbit, AlexaFluor-488 donkey anti-sheep, AlexaFluor-488 donkey anti-goat, AlexaFluor-488 donkey anti-monkey, AlexaFluor-488 goat-anti rabbit and AlexaFluor-555 donkey anti-rabbit IgG (Invitrogen).

Jensen T.B., Cheema M.U., Szymiczek A., Damkier H.H, & Praetorius J. (2015). Renal Type A Intercalated Cells Contain Albumin in Organelles with Aldosterone-Regulated Abundance. PLoS ONE, 10(4), e0124902.

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Immunofluorescence Staining of Fibroblasts

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Normal human fibroblasts or RDEB fibroblasts were plated on coverslips (Thomas Scientific, 6661-K40) in 6-well plates (Thermo Scientific, Catlog No. 140675) at 0.5 × 10⁵ cell/well. After 48 hours, cells were fixed by 4% paraformaldehyde and blocked by 3% fetal bovine serum in 0.1% Triton X-100 for half hour at room temperature. Primary antibodies used were Collagen VII (Sigma Prestige, HPA042420, Rabbit, 1:50 dilution), Thrombospondin-1 (Santa Cruz, sc-59887, mouse, 1:50 dilution), SEC23 (Abcam, ab99552, goat, 1:100 dilution), SEC31 (Santa Cruz, sc-376587, mouse, 1:50 dilution), pSMAD3 S423/S425(Rockland, 600-401-919, Rabbit, 1:50 dilution), Calreticulin (Life Span Bioscience, LS-B5223-125, Sheep, 1:50 dilution) and Collagen I (SouthernBiotech, 1310-01, goat, 1:50). Cells with primary antibodies were incubated for 2 hours at room temperature. Secondary antibodies, Alexa Fluor 594 goat anti-rabbit (1:800) (Invitrogen, Eugene, OR), Alexa Fluor 488 goat anti-mouse (1:250) (Invitrogen, Eugene, OR), Alexa Fluor 647 donkey anti-goat (Invitrogen, Eugene, OR) and Alexa Fluor 488 donkey anti-sheep (1:250) (Invitrogen, Eugene, OR), were applied for 1 hour at room temperature. Coverslips were mounted on the slides with DAPI Fluoromount-G (SouthernBiotech, #0100-20) and analyzed by confocal microscopy (Nikon A1R Microscope).

Cao Q., Tartaglia G., Alexander M., Park P.H., Poojan S., Farshchian M., Fuentes I., Chen M., McGrath J.A., Palisson F., Salas-Alanis J, & South A.P. (2022). Collagen VII maintains proteostasis in dermal fibroblasts by scaffolding TANGO1 cargo. Matrix biology : journal of the International Society for Matrix Biology, 111, 226-244.

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Immunofluorescence Staining of Fibroblasts

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