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Alexa488 and alexa594 conjugated secondary antibodies

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Alexa488 and Alexa594 conjugated secondary antibodies are fluorescently labeled antibodies used in various immunodetection techniques. These antibodies are designed to bind to the primary antibodies targeting specific proteins or antigens, allowing for visualization and detection of the target molecules. The Alexa488 and Alexa594 dyes provide bright and photostable fluorescent signals that can be detected using appropriate fluorescence microscopy or flow cytometry systems.

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Lab products found in correlation

Dm2000, by Leica (1 mentions) Vectashield with dapi, by Vector Laboratories (1 mentions) Las version 3, by Leica (1 mentions) Vimentin, by Progen Biotechnik (1 mentions) Cryotome, by Leica (1 mentions) Ba 9400, by Vector Laboratories (1 mentions) Mca1957ga, by Bio-Rad (1 mentions) Ab108539, by Abcam (1 mentions) Abc elite kit, by Vector Laboratories (1 mentions) Ba 2001, by Vector Laboratories (1 mentions) Ba 1000, by Vector Laboratories (1 mentions) Metamorph software, by Molecular Devices (1 mentions) Vectashield mounting solution, by Vector Laboratories (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Rat anti cd31, by BD (1 mentions) Biotinylated secondary antibody, by Vector Laboratories (1 mentions) Mms 435p, by Merck Group (1 mentions) Sc 7819, by Santa Cruz Biotechnology (1 mentions) Anti f4 80, by Bio-Rad (1 mentions) Hoechst 33432, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Alexa594-conjugated secondary antibodies (9 citations) Alexa488-and Alexa594-conjugated antibodies (2 citations)

8 protocols using alexa488 and alexa594 conjugated secondary antibodies

1

Histological and Immunohistological Characterization of Valve Tissue

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Frozen tissue sections of 5 μm thickness were fixed with 4% formalin and subjected to histological and immunohistological staining. Hematoxylin and eosin (H&E) staining was assessed for orientation and tissue integrity and Movat pentachrome staining was used to assess distribution of collagen, glycosaminoglycans, and elastic fibers within the valvular tissue. Biomineralization was confirmed via Alizarin red and von Kossa staining. Staining procedures were performed as previously described [20 (link),21 (link)].
Immunohistological staining was performed as previously described [22 (link)] with antibodies targeting vimentin (Progen Biotechnik GmbH, Heidelberg, Germany) and von Willebrand factor (vWF; Agilent Technologies, Santa Clara, CA, USA). Subsequently, sections were stained with alexa488 and alexa594 conjugated secondary antibodies (Thermo Fisher Scientific). Sections were embedded with Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). Images of all sections were taken using a Leica DM 2000 and LAS version 3.8 software (Leica Microsystems, Wetzlar, Germany).
Niazy N., Barth M., Selig J.I., Feichtner S., Shakiba B., Candan A., Albert A., Preuß K., Lichtenberg A, & Akhyari P. (2021). Degeneration of Aortic Valves in a Bioreactor System with Pulsatile Flow. Biomedicines, 9(5), 462.
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2

Immunohistochemical Analysis of Synucleinopathy

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Fixed hemispheres and spinal cords were cut on a cryotome (Leica, Nussloch, Germany) at 40 μm thickness of the sections. Immunohistochemistry on free-floating sections was performed using standard protocols and the following antibodies: anti-aggregated α-synuclein (5G4, Linaris, Germany), anti-phosphorylated α-synuclein (pS129, ab51253, Abcam, United Kingdom), anti-Iba1 (ab108539, Abcam, United Kingdom), anti-tyrosine hydroxylase (TH, AB152, Millipore, Germany), anti-CD68 (MCA1957GA, Serotec, United Kingdom), biotinylated anti-mouse, anti-rat and anti-rabbit IgG (respectively, BA-1000, BA-9400 and BA-2001, Vector Laboratories, United States). The reaction was enhanced with ABC Elite kit (PK-6100, Vector Laboratories, United States) and visualized with 3, 3′-diaminobenzidine. Cresyl violet was used for counterstaining of sections immunostained with anti-Iba1 antibody. For co-localization analysis, immunofluorescence for pS129 and LC3b (Cell Signaling, Leiden, Netherlands) followed by Alexa 488- and Alexa 594-conjugated secondary antibodies (Thermo Fisher Scientific, Rockford, IL, United States) was performed.
Kallab M., Herrera-Vaquero M., Johannesson M., Eriksson F., Sigvardson J., Poewe W., Wenning G.K., Nordström E, & Stefanova N. (2018). Region-Specific Effects of Immunotherapy With Antibodies Targeting α-synuclein in a Transgenic Model of Synucleinopathy. Frontiers in Neuroscience, 12, 452.
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3

Immunofluorescence Labeling of Hippocampal Neurons

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Primary cultured hippocampal neurons were fixed with 4% paraformaldehyde (PFA)/4% sucrose for analysis of the spine or with 4% PFA/4% sucrose followed by methanol (−20°C) for immunofluorescent labelling of RAPGEF2. Neurons were incubated with primary antibodies in a GDB solution (30 mM phosphate buffer, pH 7.4, containing 0.1% gelatine, 0.3% Triton X‐100, 450 mM NaCl) at 4°C overnight and then with Alexa 488‐ and Alexa 594‐conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 h; neurons were then mounted on a glass slide with VECTASHIELD mounting solution (Vector Labs).
To quantify the immunofluorescence intensity of RAPGEF2, VGLUT1 and PSD‐95, we selected at least two dendritic segments (30 μm in length each) in the individual GFP‐positive neurons. The integrated intensity was measured at a constant threshold value using the region measurement tool of MetaMorph Software (Molecular Devices). For the analysis of dendritic spine structures, we focused on linear secondary dendritic segments of pyramidal neurons (at least 30 μm in length). Images were acquired with a TI‐RCP confocal microscope (Nikon). Z‐stack images were collected at 0.4 μm intervals and then compressed into a 2D image with maximal intensity projection.
Jang Y., Jang H., Kim G.H., Noh J., Chang K, & Lee K.J. (2021). RAPGEF2 mediates oligomeric Aβ‐induced synaptic loss and cognitive dysfunction in the 3xTg‐AD mouse model of Alzheimer’s disease. Neuropathology and Applied Neurobiology, 47(5), 625-639.
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4

Visualizing Lymphatic and Vascular Structures

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Cryosections of the tails (7 μm) were fixed for 2 min in acetone (–20°C) and for 5 min in 80% methanol (4°C), washed in PBS and incubated overnight (4°C) with a hamster anti-podoplanin antibody (clone 8.1.1, Developmental Studies Hybridoma Bank, University of Iowa) and rat anti-Meca32 antibody (1∶200, BD Pharmingen, Allschwil), or rabbit anti-LYVE-1 (1∶600, AngioBio) and rat anti-CD31 (1∶200, BD Pharmingen). The samples were then incubated for 30 min with Alexa488- and Alexa594-conjugated secondary antibodies (1∶200) and Hoechst 33342 (1∶1000; all from Invitrogen, Basel, Switzerland). Regular hematoxylin staining was used to stain paraffin sections (8 μm) of the tail.
Blum K.S., Karaman S., Proulx S.T., Ochsenbein A.M., Luciani P., Leroux J.C., Wolfrum C, & Detmar M. (2014). Chronic High-Fat Diet Impairs Collecting Lymphatic Vessel Function in Mice. PLoS ONE, 9(4), e94713.
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5

Immunohistochemistry for Neuronal Markers

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We used the following antibodies: ChAT (Chemicon AB144P), Otx2 (R&D systems goat, catalog # AF1979), Tuj1 (Covance MMS-435P), pH3 (Millipore 06-570), parvalbumin (Millipore MAB1572), somatostatin (Santa Cruz sc-7819).
Cryosections were rinsed in PBS, blocked in 10% normal serum/PBST (1x PBS, 0.1% Triton X-100), incubated in primary antibody overnight (4° C), washed in PBST, incubated in secondary antibody 1–3 hours (room temperature), and washed in PBS. For fluorescent detection, we used Alexa 488- and Alexa 594-conjugated secondary antibodies (Invitrogen). For colorimetric detection, biotinylated secondary antibodies (Vector) were used with the ABC (Vector)/DAB detection method.
For ChAT IHC, antigen retrieval was achieved by incubating slides in 2.94g/L trisodium citrate dehydrate, 0.05% Tween-20, pH 6.0 for 15 minutes at 90° C. Blocking and antibody incubations were done in 1% BSA in PBST. Sections were incubated two days at 4° C with primary antibody, and signal was amplified with biotinylated anti-goat (Vector) prior to fluorescent detection with streptavidin-594 (Invitrogen).
For OTX2 IHC, we modified the IHC protocol according to the recommendations of Yuki Muranishi in the Furakawa lab (Osaka, Japan). Briefly, antigen retrieval was achieved as for ChAT IHC, and samples were blocked in 4% donkey serum in PBST.
Hoch R.V., Lindtner S., Price J.D, & Rubenstein J.L. (2015). OTX2 Transcription Factor Controls Regional Patterning Within The Medial Ganglionic Eminence (MGE) And Regional Identity Of The Septum. Cell reports, 12(3), 482-494.
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6

Immunofluorescence Analysis of Tumor Sections

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Tumors were also frozen, and embedded in optimal cutting medium (OCT 4583; Sakura Finetek) for immunofluorescence analysis. Sections were fixed in 4% paraformaldehyde, blocked with horse serum containing 2.5% of fraction V for 1 hour, and then stained with primary antibodies at 4°C overnight: anti–IL6 (1:200, Abcam), or anti-F4/80 (1:200, AbD Serotec). After two washes with PBS, sections were stained with Alexa488- and Alexa594-conjugated secondary antibodies (1:500, Invitrogen) for 30 minutes. Nuclei were counterstained with Hoechst 33432 (Invitrogen) for 5 minutes. Slides were mounted and examined with a LSM710 laser-scanning confocal microscope (Zeiss).
Nagahashi M., Yamada A., Katsuta E., Aoyagi T., Huang W.C., Terracina K.P., Hait N.C., Allegood J.C., Tsuchida J., Yuza K., Nakajima M., Abe M., Sakimura K., Milstien S., Wakai T., Spiegel S, & Takabe K. (2018). Targeting the SphK1/S1P/S1PR1 Axis That Links Obesity, Chronic Inflammation, and Breast Cancer Metastasis. Cancer research, 78(7), 1713-1725.
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7

Histological Analysis of Heart Sections

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Histological analyses of the heart sections were conducted as described previously [23] (link). The heart specimens were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned at 6-μm thickness. For immunofluorescent staining, leukocytes were stained with anti-Gr-1 antibody (BD Biosciences). Alexa 488- and Alexa 594-conjugated secondary antibodies (Life Technologies Japan, Tokyo, Japan) were used. Nuclei were stained with DAPI.
Kitano K., Usui S., Ootsuji H., Takashima S.I., Kobayashi D., Murai H., Furusho H., Nomura A., Kaneko S, & Takamura M. (2014). Rho-Kinase Activation in Leukocytes Plays a Pivotal Role in Myocardial Ischemia/Reperfusion Injury. PLoS ONE, 9(3), e92242.
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8

Immunofluorescence Microscopy of Trypanosomes

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1 × 107 BSF cells were harvested (1500 g, 10 min, RT) and resuspended in 1 ml HMI-9 and fixed in 2% formaldehyde for 5 min at RT. The fixed cells were washed three times with PBS and resuspended in 500 μl PBS. A total of 100 μl of cells were added to poly-L-lysine-coated slides and allowed to settle for 20 min at RT. Attached trypanosomes were then permeabilized with 0.2% Igepal CA-630 in PBS for 5 min at RT. After washing twice with PBS cells were blocked with 1% BSA in PBS for 1 h at 37°C. TelAP1 mouse monoclonal IgG and TbTRF rat monoclonal IgG were used to detect TelAP1 and TbTRF, respectively. The primary antibodies were applied for 1 h at RT. After three washes with PBS, Alexa488- and Alexa594-conjugated secondary antibodies (Life Technologies) and Hoechst to stain DNA were applied for 45 min at RT. After three washing steps with PBS, cells were mounted in Vectashield (Vecta Laboratories Inc.), and images were captured by using an IMIC microscope (TILL Photonics, Gräfelfing, Germany). Deconvolution was carried out using the Huygens Essential software 4.1 (Scientific Volume Imaging).
Reis H., Schwebs M., Dietz S., Janzen C.J, & Butter F. (2018). TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes. Nucleic Acids Research, 46(6), 2820-2833.
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