Alexa fluor 488 donkey anti rabbit

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Japan

Alexa Fluor 488 donkey anti-rabbit is a fluorescently labeled secondary antibody used for detection and visualization in various immunoassay techniques. It is designed to bind to rabbit primary antibodies, allowing for sensitive and specific target detection.

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245 protocols using alexa fluor 488 donkey anti rabbit

Collagen Immunofluorescence in Sectioned Femurs

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Sectioned femur samples (n = 6/group) (pre-treated in xylene for 10 min to clear away the paraffin) were incubated with testicular hyaluronidase for 30 min to expose collagen epitopes. The samples were immunofluorescently labeled for 1 h at room temperature either with rabbit polyclonal IgG collagen type II (10 μg/ml; ab34712; Abcam, UK) followed by Alexa 488 donkey anti rabbit IgG (2 μg/ml; Invitrogen, Eugene, OR, USA) or rabbit polyclonal collagen IX (10 μg/ml; Abcam) followed by Alexa 647 goat anti-rabbit IgG (2 μg/ml; Invitrogen) or Rabbit (Rb) pAb collagen X (10 μg/ml; ab58632; Abcam) followed by Alexa fluor 488 donkey anti-rabbit (2 μg/ml; Invitrogen) or rabbit polyclonal IgG osteocalcin (10 μg/ml; Santa Cruz Biotechnology, CA, USA) followed by Alexa fluor 488 donkey anti-rabbit (2 μg/ml; Invitrogen). Antibodies were diluted in 3% (w/v) bovine serum albumin (BSA). The nuclear stain bisbenzimide (Sigma Aldrich; Hoechst dye No. 33258, dissolved in H₂O) was administered for 5 min and coverslips were mounted on slides using Airvol as described previously [21 (link), 22 (link)]. Images were taken (n = 8 image sections/sample) on the Zeiss 780 confocal with a 20× objective (0.75NA, Beam Splitter (MBS) 458/514/561/633, 5% laser output, and (MBS) 405, 2% laser output). Images were quantified using ImageJ (NIH, Bethesda).

Akkiraju H., Srinivasan P.P., Xu X., Jia X., Safran C.B, & Nohe A. (2017). CK2.1, a bone morphogenetic protein receptor type Ia mimetic peptide, repairs cartilage in mice with destabilized medial meniscus. Stem Cell Research & Therapy, 8, 82.

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Immunohistochemistry of Neural Cell Markers

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Tissue was sectioned at 10 μm and following deparaffinization (3 day only) samples underwent antigen retrieval in Tris EDTA buffer at pH 9 for 15 min at 95°C. A standard IHC protocol was followed [17 ], in brief, samples were permeabilized in 0.1% Triton X-100 for 7 minutes, rinsed, then incubated in 100% FBS for 1 hour, rinsed, then incubated in primary antibody overnight at 4°C, rinsed three times, then incubated in secondary antibody for one hour at room temperature, rinsed three times, then incubated in 10 μM DAPI for 5 min, rinsed, and mounted with Prolong Gold (Thermo-Fisher Scientific). Antibodies used are as followed: NSC marker Nestin 1:20 (DSHB RAT-401). Neuronal marker βIII tubulin 1:400 (BioLegend, 801201), neutrophil marker myeloperoxidase (Thermofisher) 1:200, macrophage marker ED-1 clone (Sigma Aldrich) 1:150 with 1:500 secondary Alexa Fluor 488 donkey anti-rabbit and Alexa Fluor 647 goat anti-mouse (ThermoFisher Scientific). Slides exposed to the whole process except for primary antibodies were used as negative controls throughout. Imaging was performed using a Nikon A1 confocal system or Olympus FV1000 with gain and laser power consistent for each fluorescent secondary antibody used. ImageJ was used to threshold images and quantify positive staining normalized to DAPI.

Barkla B.J., Vera-Estrella R., Maldonado-Gama M, & Pantoja O. (1999). Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated. Plant Physiology, 120(3), 811-820.

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Immunofluorescence Labeling of Cultured Cells

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Cells were grown to 80% confluence on cover slips in 12-well plates prior to fixation with 0.5 ml 4% methanol-free formaldehyde (Thermo Fisher Scientific) for 4 min at room temperature. Permeabilisation was achieved using 0.5 ml 100% ice-cold ethanol and incubation at −20°C overnight. Ethanol was removed and 1 ml wash buffer (1x TBS, 0.2% Triton X100 and 0.04% SDS, Sigma-Aldrich was added to the wells for 5 min. Cells were blocked in 1 ml blocking buffer (3% bovine serum albumin diluted in 1xTBS) for 1 h at room temperature. Cover slips were incubated with 60 μl primary antibody diluted in blocking buffer for 1 h at room temperature. Three washes were performed with 1 ml wash buffer for 5 min each. Secondary antibodies (Alexa Fluor 488 donkey anti-rabbit or Alexa Fluor 568 donkey anti-mouse, Thermo Fisher Scientific) were diluted in blocking buffer and 60 μl was added to each cover slip for 1 h at room temperature in the dark. Cells were washed as before prior to mounting the coverslips with mounting medium containing DAPI (Vectashield) on microscope slides (ThermoScientific) sealed with nail varnish at the edges. Slides were stored at 4°C in the dark until analysis. Images were analysed on the Zeiss Observer Z1 Microscope using a 40X oil objective or a 20X objective. Images were analysed using ImageJ software.

Taylor C., Mannion D., Mirand a F., Karaminejadranjbar M., Herrero-Gonzalez S., Hellner K., Zheng Y., Bartholomeusz G., Bast RC J.r, & Ahmed A.A. (2017). Loss of PFKFB4 induces cell death in mitotically arrested ovarian cancer cells. Oncotarget, 8(11), 17960-17980.

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Quantifying NF-κB p65 Activation in Murine Livers

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IP LPS-exposed and control livers from p7 and adult mice were fixed with 4% paraformaldehyde, paraffin-embedded, and stained against NF-κB p65 subunit. Briefly, after antigen retrieval (antigen unmasking solution, H-3301, Vector Laboratories, Burlingame, CA, USA), permeabilization (0.5% Triton X), and quenching with 100 mM glycine and 0.5% Pontamine Sky Blue (Chicago Sky Blue 6B, C8679-25G, Sigma-Aldrich, St. Louis, MO, USA), 5 μm sections were blocked with Sea Blocking (#37527, ThermoFisher Scientific, Waltham, MA) for 40 min, and Fc Receptor Blocking (NB309-15, Innovex Biosciences, Richmond, CA, USA) for 30 min. Sections were subsequently incubated with anti-p65 antibody (1:100, NF-κB p65 XP®, #8242, Cell Signaling, Danvers, MA, USA) at 4°C overnight. The following day, sections were incubated with secondary antibody (1:200, Alexa Fluor 488 donkey anti-rabbit, A-21206, ThermoFisher Scientific, Waltham, MA) for 1 h at room temperature. Finally, sections were mounted with DAPI (1:1000, D9542, Sigma-Aldrich, St. Louis, MO, USA) and imaged with an IX83 microscope and DP80 camera using CellSens software (Olympus Life Science, Waltham, MA, USA).

Zarate M.A., Nguyen L.M., De Dios R.K., Zheng L, & Wright C.J. (2020). Maturation of the Acute Hepatic TLR4/NF-κB Mediated Innate Immune Response Is p65 Dependent in Mice. Frontiers in Immunology, 11, 1892.

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GFP Immunostaining of Retinal Tissue

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After recording, retinas were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in 1X PBS for 30 min-1 hr at room temperature (RT) and then washed 3X20 min in 1X PBS. Retinas were then placed in blocking solution containing 5% donkey serum (Sigma) in 0.3% Triton-X PBS (PBS_TX) for 1 hr at RT. Then, retinas were incubated in primary antibody solution containing rabbit anti-GFP (1:1000, Thermo) in 2% donkey PBS_TX for 3 days at 4 C and washed for 3X20 min in 1X PBS, and then incubated in secondary antibody solution including Alexa Fluor 488 donkey anti-rabbit (1:1000, Thermo) in 2% donkey PBS_TX for 2 hrs at RT. Alexa Fluor 546-conjugated streptavidin (1:500, Thermo) was included in both primary and secondary antibody solution. Retinas were washed for 3X20 min in 1X PBS and then mounted using Fluoromount aqueous mounting medium (Sigma).

Lee S.K., Sonoda T, & Schmidt T.M. (2019). M1 Intrinsically Photosensitive Retinal Ganglion Cells Integrate Rod and Melanopsin Inputs to Signal in Low Light. Cell reports, 29(11), 3349-3355.e2.

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Immunohistochemical Staining of Brain Slices

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Brain slices (30 μm) were prepared as previously described [19 (link)]. Sections were mounted on poly-D-lysine coated slides (Thermo Fisher), antigen retrieval with sodium citrate buffer was performed, except when staining with the TOC1 antibody, and the slides were blocked with PBS containing 5% BSA and 0.1% tween 20 for 1 h at room temperature. The sections were incubated with primary antibody in 5% BSA in PBS overnight at 4°C. The next day, slices were incubated for 1 h at room temperature with Alexa Fluor^™-conjugated secondary antibody including Alexa Fluor^™ 594 donkey-anti-rabbit, Alexa Fluor^™ 488 donkey-anti-rabbit, or Alexa Fluor^™ 647 donkey-anti-rabbit (Thermo Fisher Scientific). Alternatively, they were labeled using the MOM kit (BMK-2202, Vector laboratories), followed by three washes with PBS and labeling with Streptavidin Alexa Fluor^™ 488 or 647 (Thermo Fisher Scientific). The brain sections were coverslipped with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, P36961). The slides were imaged using a Nikon A1R HD laser scanning confocal microscope and recorded by NIS-Elements (Version 5.11) software. Resulting images were pseudocolored for illustration purposes.

Lin H., Sandkuhler S., Dunlea C., King D.H, & Johnson G.V. (2023). BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1. bioRxiv.

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Optimized Confocal Imaging of Transfected Cells

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Transfected cells were fixed with 4% paraformaldehyde for 20 min at RT, washed with PBS, and mounted with ProLong Antifade Mountant (Thermo Fisher Scientific, Rockford, IL, USA) for imaging. Samples were visualized using a Zeiss LSM 700 confocal microscope using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective. Zeiss ZEN 2010 program was used to control imaging specifications. The gap junction area was determined using ImageJ by tracing the gap junction area with a free hand tool followed by quantification using the measure tool. ImageJ software was also used to analyze co-localization data.
In the case of immunofluorescence, transfected cells were fixed with ice-cold 100% methanol for 10 min at RT. Cells were blocked using PBS supplemented with 2% BSA for 1 h at RT. Primary antibody, rabbit anti-HIS (Bethyl Laboratories Inc., Montgomery, TX, USA) at 1:1000 concentration, was diluted in PBS with 0.1% BSA and applied to cells for 1 h at RT. Cells were then incubated in the secondary antibody solution containing 2 μg/mL of Alexa Fluor 568 goat anti-mouse (Thermo Fisher Scientific, Rockford, IL, USA) and 2 μg/mL of Alexa Fluor 488 donkey anti-rabbit (Thermo Fisher Scientific, Rockford, IL, USA) in PBS with 0.1% BSA for 1 h at RT. Cells were washed in PBS and mounted with FluoroshieldTm (Sigma-Aldrich Chemie GmbH, Munich, Germany).

Kotova A., Timonina K, & Zoidl G.R. (2020). Endocytosis of Connexin 36 is Mediated by Interaction with Caveolin-1. International Journal of Molecular Sciences, 21(15), 5401.

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Quantitative Immunofluorescence Analysis of HDAC4 Phosphorylation

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Immunofluorescence was performed in μ-Slide 8 Well chambers (#80826, Ibidi). 25,000 HMECs per well were seeded in EBM+0.1% BSA for the indicated periods. Then, cells were fixed in 2% PFA for 10 min and washed twice with PBS. To remove PFA PBS+2% glycine was added for 10 min and cells were washed twice with PBS afterwards. 0.05% Triton X-100 in PBS was used to permeabilize the cells for 10 min. PBS+3% BSA was added for 30 min to reduce unspecific binding. Phospho HDAC4 (#ab39408, Abcam) was diluted in PBS+3% BSA (1:100) and incubated over night at 4 °C. Cells were washed three times with PBS+0.3% Tween and once with PBS. Secondary antibody AlexaFluor488 donkey anti-rabbit (#A21206, Thermo Fisher Scientific) 1:500 in PBS+3% BSA was added for 30 min at room temperature. Cells were stained with DAPI for 10 min and washed three times with PBS+0.3% Tween and once with PBS. Images were taken with a confocal microscope LSM 510 Meta and analyzed with ImageJ.

Schader T., Löwe O., Reschke C., Malacarne P., Hahner F., Müller N., Gajos-Draus A., Backs J, & Schröder K. (2020). Oxidation of HDAC4 by Nox4-derived H2O2 maintains tube formation by endothelial cells. Redox Biology, 36, 101669.

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Immunofluorescence Analysis of Microglial Markers

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After treatment, the BV2 cells were fixed for 15 min in 4% paraformaldehyde, permeabilized for 7 min with 0.1% Triton X-100, and then blocked for 30 min with 1% BSA. The cells were incubated for 1 h at RT with mouse anti-CD86 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, sc-28347, 1:250), mouse anti-CD68 (Santa Cruz Biotechnology Inc., sc-20060; 1:250), a rabbit anti-IL-10 antibody (Abbiotec, 250713; 1:200), a rabbit anti-TNF-α antibody (Novus Biologicals, NB600-587; 1:100), or a rabbit anti-α7 nAchR (Abcam, ab216485; 1:500). After washing in PBS three times for 5 min each, the cells were incubated for 1 h at RT in the dark with the appropriate fluorescent-labelled secondary antibodies (Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific), Alexa Fluor 546 donkey anti-rabbit (Thermo Fisher Scientific), or Alexa Fluor 488 donkey anti-rabbit (Thermo Fisher Scientific)). Finally, for nuclear staining and the stabilization of fluorescent signals, the slides were covered in mounting medium (Fluoroshield with DAPI; Sigma-Aldrich, Milan, Italy) and secured with a coverslip. Fluorescence images were captured using a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss LSM 700, Jena, Germany).

Cantone A.F., Burgaletto C., Di Benedetto G., Pannaccione A., Secondo A., Bellanca C.M., Augello E., Munafò A., Tarro P., Bernardini R, & Cantarella G. (2024). Taming Microglia in Alzheimer’s Disease: Exploring Potential Implications of Choline Alphoscerate via α7 nAChR Modulation. Cells, 13(4), 309.

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Immunofluorescence Staining of Nephrocytes

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For immunofluorescence, nephrocytes were dissected, fixed for 20 min in PBS containing 4% paraformaldehyde, and stained according to the standard procedure. The following primary antibodies were used: rabbit anti-sns (Bour et al., 2000 (link)) (1:300, gift from S Abmayr) and guinea pig anti-Kirre (Galletta et al., 2004 (link)) (1:200, gift from S Abmayr). Other antibodies used were rabbit anti-Rab5 (ab18211, abcam, 1:100), mouse anti-Rab7 (Rab7, DSHB 1:100), mouse anti-Myc (9E10; DSHB, 1:100), mouse anti-c-Myc (sc-40; Santa Cruz Biotechnology 1:100), and rabbit anti-RAB11 (#5589S; Cell Signaling Technology, 1:100). The following secondary antibodies were used: Alexa Fluor 488 donkey anti-rabbit (#A-21206, Thermofisher, 1:200), Alexa Fluor 488 donkey anti-mouse (#A32766, Thermofisher, 1:200), Alexa Fluor 568 donkey anti-rabbit (#A10042, Thermofisher, 1:200), Alexa Fluor 568 donkey anti-mouse (#A10037, Thermofisher, 1:200), Alexa Fluor 568 goat anti-guinea pig (#11075, Thermofisher, 1:200).
Apoptotic cells were visualized using the In Situ Cell Death Detection Kit (#11684795910, Sigma/Roche) according to the manufacturer’s instructions. For imaging, a Zeiss LSM 880 laser scanning microscope was used. Image processing was done by ImageJ and GIMP software.

Lang K., Milosavljevic J., Heinkele H., Chen M., Gerstner L., Spitz D., Kayser S., Helmstädter M., Walz G., Köttgen M., Spracklen A., Poulton J, & Hermle T. (2022). Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes. eLife, 11, e79037.

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