Alexa 594

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Canada, Switzerland, Japan, Germany, France, Spain

Alexa Fluor 594 is a fluorescent dye produced by Thermo Fisher Scientific. It is designed for use in a variety of biological applications, including flow cytometry, immunohistochemistry, and fluorescence microscopy. The dye has an excitation maximum at 590 nm and an emission maximum at 617 nm, making it compatible with common fluorescence detection systems.

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Isolectin B4 (lectin) (Alexa Fluor 594 – I21413, Alexa Fluor 594-Phalloidin conjugate Alexa Fluor 594-conjugated goat anti-rat Alexa-Fluor 594-nm conjugated secondary antibodies ChromaTide Alexa Fluor 488-5 or 594-5-dUTP Alexa Fluor 594 anti-goat IgG Alexa-Fluor 594 conjugate anti-mouse secondary antibody Anti-Mouse Alexa® Fluor 488 and anti-rabbit Alexa® Fluor 594 conjugated secondary antibody Alexa 594-conjugated anti-rabbit IgG Rabbit Alexa Fluor 594

538 protocols using alexa 594

Immunostaining of GABA Neuron Markers

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Eight-week-old Pin1+/+ and Pin1−/− littermates (for each genotype, n=3) were anesthetized and perfused transcardially with 0.1 M phosphate buffer, pH 7.4 (PB). Brains were quickly remove from the skull and frozen with isopentane cooled to −40°C with liquid nitrogen. Ten to twelve μm thick cryostat sections were collected on Superfrost glass slides and further processed for immunostaining for combined detection of VGAT and GABAA γ2 or VGAT and gephyrin. Briefly, cryostat sections were fixed by immersion in 2% paraformaldehyde, and mildly treated with pepsin as antigen-retrieval procedure, and then incubated for 48 hours with different combination of primary antibodies. Secondary antibody staining was performed for 1 h at room temperature using anti-isotypic fluorophore conjugated antibodies Alexa-488 and Alexa-594 at dilutions of 1:1000 (Molecular Probes).
Hippocampal neurons grown on glass coverslips were fixed with 4% paraformaldehyde and 4% sucrose in PBS. Unspecific binding was blocked by incubation with 10% normal goat serum (NGS) in PBS. Primary and secondary antibodies were diluted in 5% NGS/PBS. Secondary antibodies included anti-isotypic fluorophore conjugated antibodies Alexa-488, Alexa-594 and streptavidin-Alexa 405 at dilutions of 1:1000 (Molecular Probes).

Antonelli R., Pizzarelli R., Pedroni A., Fritschy J.M., Del Sal G., Cherubini E, & Zacchi P. (2014). Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction. Nature Communications, 5, 5066.

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Immunostaining of GABA receptors

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Eight-week-old Pin1+/+ and Pin1−/− littermates (for each genotype, n=3) were anaesthetized and perfused transcardially with 0.1 M phosphate buffer, pH 7.4 (PB). Brains were quickly removed from the skull and frozen with isopentane cooled to −40 °C with liquid nitrogen. Ten to 12-μm thick cryostat sections were collected on Superfrost glass slides and further processed for immunostaining for combined detection of VGAT and GABAA γ2 or VGAT and gephyrin. Briefly, cryostat sections were fixed by immersion in 2% paraformaldehyde, and mildly treated with pepsin as antigen-retrieval procedure, and then incubated for 48 h with different combination of primary antibodies. Secondary antibody staining was performed for 1 h at room temperature using anti-isotypic fluorophore-conjugated antibodies Alexa-488 and Alexa-594 at dilutions of 1:1,000 (Molecular Probes).
Hippocampal neurons grown on glass coverslips were fixed with 4% paraformaldehyde and 4% sucrose in PBS. Unspecific binding was blocked by incubation with 10% normal goat serum in PBS. Primary and secondary antibodies were diluted in 5% normal goat serum/PBS. Secondary antibodies included anti-isotypic fluorophore conjugated antibodies Alexa-488, Alexa-594 and streptavidin-Alexa 405 at dilutions of 1:1,000 (Molecular Probes).

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Immunostaining analysis of hiPSCs and AECs

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Undifferentiated hiPSCs and AECs were rinsed with PBS and fixed with 4% paraformaldehyde (Alfa Aesar) for 20 min at room temperature. Cells were permeabilized with 0.5% saponin (Sigma) and blocked with 10% normal donkey serum (Jackson Immuno., USA) diluted with 1% BSA in PBS. The following primary and secondary antibodies were used: rabbit anti-OCT4 (1:200, BD Pharmingen), rabbit anti-NKX2.1 (1:250, Abcam), mouse anti-CPM (1:500, Abcam), mouse anti-EPCAM (1:400, Santa Cruz), Alexa 594 (Invitrogen) donkey anti-mouse IgG(H + L), Alexa 488 (Invitrogen) donkey anti-rabbit IgG(H + L) and Alexa 594 (Invitrogen) donkey anti-rabbit IgG(H + L). Nuclei were counterstained with Fluoroshield with DAPI (Sigma) for 5 min, and fluorescent images were captured with a fluorescence microscope (IX-51, Olympus, JAPAN).

Heo H.R., Kim J., Kim W.J., Yang S.R., Han S.S., Lee S.J., Hong Y, & Hong S.H. (2019). Human pluripotent stem cell-derived alveolar epithelial cells are alternatives for in vitro pulmotoxicity assessment. Scientific Reports, 9, 505.

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Immunostaining of Embryonic Stem Cells

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Each stage of embryos without zona pellucida was fixed in 4% paraformaldehyde for 15 minutes at room temperature. The fixed samples were permeabilized using 1% Triton ×‐100 for 1 hours at room temperature and then washed three times with phosphate‐buffered saline (PBS). The embryos were blocked using 10% goat serum or donkey serum in PBS for 1 hours at room temperature. Samples were stained with anti‐SOX2 (5 μg/mL), anti‐NANOG (1 μg/mL), anti‐OCT4 (1 μg/mL) and anti‐SOX17 (1 μg/mL) antibodies in PBS containing 10% goat serum or donkey serum at 4°C overnight (Table S2). After washing three times in washing solution (PBS with 0.2% Tween‐20 and 1% BSA for 10 minutes), the embryos were incubated with goat anti‐rabbit Alexa 594 (Invitrogen) or donkey anti‐rabbit Alexa594 (Invitrogen) antibodies in PBS with 10% goat serum or donkey serum at RT for 1 hours. All samples were washed three times with washing solution after secondary antibody treatment. Immunostained embryos were mounted on a slide glass with Prolong Gold with DAPI (Invitrogen) and cured for more than 24 hours. We have described the list of antibodies in Table S2. Images of stained cells were captured using an inverted fluorescence microscope and processed by the ImageJ program.

Lee M., Choi K., Oh J., Kim S., Lee D., Choe G.C., Jeong J, & Lee C. (2021). SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre‐implantation embryo development. Cell Proliferation, 54(8), e13097.

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Immunocytochemical Characterization of RUES2-CMs

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RUES2-CMs were first seeded on the ibidi µ-Slide 8-well chambered slides (Martinsried, Germany). The cells were fixed with 4% paraformaldehyde prior to being blocked with 5% bovine serum albumin (BSA). The cells were stained with α-actinin (1:200; Abcam, Cambridge, UK), cardiac troponin T (cTnT) (1:200; Abcam), and PD-L1 (1:100; Proteintech Group Inc., Chicago, IL, USA) antibodies and incubated overnight at 4 °C. The cells were incubated with appropriate secondary antibodies (Alexa-488 and Alexa-594; 1:500; Invitrogen). For CD4⁺ and CD8⁺ T-lymphocytes, cells were added to cytospin slide chambers with blotters and glass slides attached. The cells were then centrifuged on a glass slide using a cytocentrifuge. The staining process was done by adding the PD-1 antibody (1:100; Proteintech Group Inc., Rosemont, IL, USA) and then appropriate secondary antibodies (Alexa-488 and Alexa-594; 1:500; Invitrogen). 4’,6-diamidino-2-phenylindole (DAPI) was utilized to observe cells’ nuclei.

Tay W.T., Fang Y.H., Beh S.T., Liu Y.W., Hsu L.W., Yen C.J, & Liu P.Y. (2020). Programmed Cell Death-1: Programmed Cell Death-Ligand 1 Interaction Protects Human Cardiomyocytes Against T-Cell Mediated Inflammation and Apoptosis Response In Vitro. International Journal of Molecular Sciences, 21(7), 2399.

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Immunofluorescence Analysis of Spinal Cord in Diabetic Mice

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Immunohistochemistry assays were performed on paraffin-embedded 7-µM-thick microtome sections of lumbar (L4–L6) spinal cords that were removed from diabetic mice on day 7 after STZ administration. Section preparation and immunofluorescence staining were performed as described by Chmielarz et al. (43 (link)). Briefly, after deparaffinization and subsequent antigen retrieval (microwave method with citrate buffer), sections from STZ-treated mice were incubated for 30 min in 5% normal pig serum (Vector Labs) in PBST buffer (0.2% Triton X-100 in PBS). Sections were incubated overnight at 4°C with the following primary antibodies: anti-CCL3 (1:50, ThermoFisher Scientific), anti-CCL9 (1:50, Bioss), anti-CCR1 (1:50, Novus), anti-CCR5 (1:50, Novus), anti-NeuN (1:200, Millipore), anti-Iba1 (1:50, Abcam, UK), and anti-GFAP (1:500, Millipore). Antigen-bound primary antibodies were visualized with anti-rabbit Alexa-488, anti-mouse Alexa-594, or anti-chicken Alexa-594 (1:100; Invitrogen) secondary antibodies. Stained sections were examined and photographed under a fluorescence microscope (Nikon Eclipse 50i). The dorsal part of the lumbar spinal cord was visualized in representative images.

Rojewska E., Zychowska M., Piotrowska A., Kreiner G., Nalepa I, & Mika J. (2018). Involvement of Macrophage Inflammatory Protein-1 Family Members in the Development of Diabetic Neuropathy and Their Contribution to Effectiveness of Morphine. Frontiers in Immunology, 9, 494.

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Double-labeling Immunofluorescence Analysis

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Immunofluorescent histochemical procedure was applied to evaluate the double-labeling of FOS/CaMKII and FOS/GAD67 in the IC of sham and SNI mice as described in our previous study (Zhang et al., 2022 (link); Zhu et al., 2022 (link)). Briefly, mice were perfused with 0.1 mol/L PBS and 4% paraformaldehyde for fixation, and then serially cut into transverse slices with 30 μm thickness. All serial sections were then incubated with primary antisera (1:400, ab11959, Abcam, MA, United Kingdom) for 18–24 h at 4°C in 0.01 M PBS containing 1% (v/v) normal donkey serum, 0.3% (v/v) Triton X-100, 0.02% (w/v) sodium azide, and 0.12% (w/v) carrageenan (pH 7.4). Then, the sections were incubated with Alexa 488 donkey anti-rabbit (1:500, A21206, Invitrogen)/Alexa 594 donkey anti-mouse (1:500, A21203, Invitrogen, CA), and Alexa 488 donkey anti-mouse (1:500, A21202, Invitrogen)/Alexa 594 donkey anti-rabbit (1:500, A21207, Invitrogen) for 6–8 h at 4°C. If necessary, the sections were incubated with tertiary antisera for 2–4 h in 0.01 m PBS with 0.3% (v/v) Triton X-100 at 4°C. After the immunofluorescence histochemical staining, the sections were observed and images were captured using VS200 microscope (VS200, Olympus, Japan). Digital images were captured using VS200 software (Olympus).

Wang P., Si H.X., Zhu D.Y., Xing K.K., Wang J., Cao T.T., Zhao H., Liu X.D., Zhang M.M, & Chen T. (2023). Proanthocyanidins induce analgesic and anxiolytic effects in spared nerve injured mice by decreasing in vivo firing rate of pyramidal cells in the insular cortex. Frontiers in Molecular Neuroscience, 16, 1174125.

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Meiotic Chromosome Dynamics Analysis

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The preparation of meiotic nuclear spreads and immunofluorescence analysis were carried out as previously described [6 (link)]. The secondary antibodies used to detect the α-pT318 and α-pS298 phospho-specific antibodies were chicken anti-rabbit Alexa-594 [Invitrogen] and goat anti-guinea pig Alexa-594 [Invitrogen], respectively.

Penedos A., Johnson A.L., Strong E., Goldman A.S., Carballo J.A, & Cha R.S. (2015). Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1. PLoS ONE, 10(7), e0134297.

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Immunostaining of Plasmodium Sporozoites

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Sporozoites were seeded with 3% BSA/ RPMI into an 8‐well Labtek chambered cover glass. After fixation with 4% PFA for 20 min, cells were permeabilized with 0.5% TritonX for 15 min. Primary antibodies were incubated for 1 h and washed twice with PBS. Secondary antibodies together with Hoechst were incubated for 1 h. Cells were washed twice with PBS and observed under the microscope. Images were either taken on a Zeiss CellObserver widefield (63×) or Nikon/PerkinElmer spinning disc (100×) microscope. Image processing was performed with ImageJ. Antibodies: rabbit anti‐GFP for IFA 1/40 (abfinity 0.4 µg/µl), mouse anti CSP (mAb 3D11, 1/100, Yoshida et al, 1980 (link)), goat anti‐mouse Alexa 594 1/1,000 (Invitrogen 2 mg/ml), goat anti‐rabbit Alexa 594 1/1,000 (Invitrogen 2 mg/ml). For STED imaging, anti‐mouse Atto 594 1/300 (Sigma) and anti‐rabbit Atto 647N 1/300 (Sigma) was used. The staining of sporozoites with antibodies or SiR‐tubulin was performed as described previously (Spreng et al, 2019 (link)).

Kehrer J., Formaglio P., Muthinja J.M., Weber S., Baltissen D., Lance C., Ripp J., Grech J., Meissner M., Funaya C., Amino R, & Frischknecht F. (2022). Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission. EMBO Reports, 23(7), e54719.

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Immunostaining of Neuronal Markers

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Fourteen hours after 1 μM ICT treatment was performed (as previously described), the cells cultured on cover glass slides in 24-well plates (1 × 10⁵/well) were fixed with 4% paraformaldehyde, washed in 0.1 M PBS for 20 min and permeabilized by 0.3% Triton X-100 for 10–15 min. They were then washed 3 times using PBS for 5 min and then nonspecific antigens were blocked using 3% bovine serum albumin (BSA) for 30 min at room temperature. The cells were subsequently immunostained with primary antibodies against NeuN (ab177487, Abcam), GluN2A (ab227233, Abcam) and GluN2B (ab65783, Abcam). After overnight incubation at 4°C, the samples were incubated for 90 min with the following secondary antibodies: Alexa-594 or Alexa-488 IgG (Alexa-594 for red and Alexa-488 for green, 1:2000; Invitrogen, Carlsbad, CA, United States). Negative controls were prepared with identical conditions, except without primary antibodies. Images were acquired using a laser scanning confocal microscope (Carl Zeiss Microscopy GmbH).

Liu S., Liu C., Xiong L., Xie J., Huang C., Pi R., Huang Z, & Li L. (2021). Icaritin Alleviates Glutamate-Induced Neuronal Damage by Inactivating GluN2B-Containing NMDARs Through the ERK/DAPK1 Pathway. Frontiers in Neuroscience, 15, 525615.

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