Alexa 488 and alexa 568 conjugated secondary antibodies

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies are fluorescently labeled antibodies used in immunofluorescence and other detection techniques. These antibodies can be used to detect and visualize target proteins in biological samples.

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Alexa 488- and 568-conjugated secondary antibodies Alexa Fluor 488 and 568-conjugated secondary antibodies Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies Alexa 488/568 conjugated secondary antibodies Alexa-568 secondary antibodies Alexa 488 and 568 488-conjugated secondary antibodies Alexa 488 antibodies Alexa secondary antibodies Alexa 568

7 protocols using alexa 488 and alexa 568 conjugated secondary antibodies

Immunofluorescence Imaging of Cells

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Cells (3 × 10³ cells/well) were seeded in an 8-well chamber slide and treated with RA. After incubation, the cells were fixed in 3.7% formaldehyde for 15 min at room temperature and washed with PBS for 5 min. The cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then maintained in blocking buffer (3% BSA and 0.1% Triton X-100 in PBS) for 1 h. After blocking, the 8-well chamber slide was reacted with primary antibodies overnight at 4°C in a dark room. The primary antibodies were detected with Alexa 488- and Alexa 568-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, United States). The slides were washed with PBS, the nuclei were counterstained with DAPI (Sigma Chemicals, St. Louis, MO, United States), and the slides were analyzed by using a Zeiss Observer A1 microscope (Carl Zeiss, Oberkochen, Germany).

Han Y.H., Kee J.Y, & Hong S.H. (2018). Rosmarinic Acid Activates AMPK to Inhibit Metastasis of Colorectal Cancer. Frontiers in Pharmacology, 9, 68.

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Cellular Structure Regulation Assay

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Aphidicolin (2.5 μg/ml), thymidine (2 mM), brefeldin A (200 ng/ml), SP600125 (25 μM), and fibronectin (10 μg/ml) were from Sigma‐Aldrich. MK1775 (0.3 μM) was from Selleckchem. DMSO was from Carlo Erba. Hoechst 33342 was from Invitrogen. Mowiol 4‐88 was from Sigma‐Aldrich. The antibodies were from the following sources: anti‐GRASP65 (Cat# ab48533, RRID:AB_880296; Abcam) (1:3,000) and anti-pericentrin (Cat# ab4448, RRID:AB_304461; Abcam) were from Abcam; anti‐α‐tubulin (Clone DM1‐A) (RRID:AB_3067992) (1:5,000) was from Sigma-Aldrich; anti-GRASP55 was from Novus Biologicals (Cat# NBP1-00862, RRID:AB_1503318; Novus) (1:500); anti-Aurora-A was from BD Transduction Laboratories (1:200); anti-Ninein was from BioLegend (1:200) (Cat# 602802, RRID:AB_2251471; BioLegend). Alexa 488– and Alexa 568–conjugated secondary antibodies (Cat# 710369, RRID:AB_2532697, and Cat# A-11004, RRID:AB_2534072, respectively) (1:400) were from Thermo Fisher Scientific.

Mascanzoni F., Ayala I., Iannitti R., Luini A, & Colanzi A. (2024). The Golgi checkpoint: Golgi unlinking during G2 is necessary for spindle formation and cytokinesis. Life Science Alliance, 7(5), e202302469.

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Cellular and Tissue Immunofluorescence Protocol

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Cellular and tissue immunofluorescence were conducted according to a previously described method (Maejima et al., 2013 (link)). Briefly, neonatal rat cardiomyocytes plated on chamber slides (Lab-Tek) were fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-100. Mouse left ventricles were fixed in formalin and sectioned at 10-µm thickness. Tissue sections were then subjected to deparaffinization and antigen unmasking using citrate buffer and washed with PBS containing 0.3% Triton X-100. Samples were then blocked for 1 hour with PBS containing 5% bovine serum albumin in vitro experiments or with normal goat serum in vivo, and then incubated with primary antibody overnight. Cells were then incubated with Alexa 488- and Alexa 568-conjugated secondary antibodies (Invitrogen) for 3 hours. Nuclei were stained with DAPI. Images were taken using either conventional or confocal microscopy. For TUNEL staining, we used the In Situ Cell Death Detection kit (Roche) according to the manufacturer’s instructions. Masson’s trichrome, hematoxylin and eosin and wheat germ agglutinin staining were conducted as previously described (Del Re et al., 2013 (link); Maejima et al., 2013 (link)).

Sciarretta S., Zhai P., Maejima Y., Del Re D.P., Nagarajan N., Yee D., Liu T., Magnuson M.A., Volpe M., Frati G., Li H, & Sadoshima J. (2015). mTORC2 regulates cardiac response to stress by inhibiting MST1. Cell reports, 11(1), 125-136.

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Immunohistochemical Analysis of Lung Tissue

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Four percent paraformaldehyde‐fixed, paraffin‐embedded blocks of lung tissues were cut into 4‐μm sections. After deparaffinating, rehydration and retrieval, tissues were incubated with primary antibodies overnight at 4°C. Cells in 24‐hole chambers were fixed in 4% paraformaldehyde for 10 min and blocked with 0.2% Triton X‐100 dissolved in 1% BSA for 30 min. Then, cells were incubated with primary antibodies overnight at 4°C. After rinsing several times with PBS, cells were incubated with Alexa‐488‐ and Alexa‐568‐conjugated secondary antibodies (Invitrogen, United Kingdom) for 2 hr. Finally, DAPI was added to the wells for 10 min. Images were obtained with a ZEISS Imager.A1 fluorescence microscope (Gottingen, Germany). Immunofluorescence was performed using the following primary antibodies: Rabbit monoclonal antibodies, anti‐P21 and anti‐PTEN, were purchased from Abcam (United Kingdom). Mouse monoclonal antibody, anti‐SP‐C, was purchased from Santa Cruz Biotechnology (CA, USA).

Tian Y., Li H., Qiu T., Dai J., Zhang Y., Chen J, & Cai H. (2018). Loss of PTEN induces lung fibrosis via alveolar epithelial cell senescence depending on NF‐κB activation. Aging Cell, 18(1), e12858.

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Immunofluorescence Staining of Cells

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Cells were fixed in 4% paraformaldehyde for 10 min, and incubated with the indicated antibodies. The cells were incubated with Alexa 488- and Alexa 568-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature. All images were captured with a fluorescence microscope (D80i; Nikon, Tokyo, Japan). Results are representative of three independent experiments.

Noh K.T., Cho J., Chun S.H., Jang J.H., Cha G.S., Jung I.D., Jang D.D, & Park Y.M. (2015). Resveratrol regulates naïve CD 8+ T-cell proliferation by upregulating IFN-γ-induced tryptophanyl-tRNA synthetase expression. BMB Reports, 48(5), 283-288.

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Immunofluorescence Analysis of p53 and Lamin A/C

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Cells were plated into 2 well chamber slides (Nunc™ Lab-Tek™ II) at a density of 1 × 10⁵ cells/mL. After one day plating, cells were treated with DMSO or 40μM Atorvastatin for 24 h at 37°C. Cells were washed with PBS, fixed by 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at room temperature, and blocked by 3% BSA in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Cells were incubated overnight with anti-p53 (FL-393) antibody (1:50, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-lamin A/C (4C11) antibody (1:200, Cell Signaling Technology), followed by incubation with Alexa-568 and Alexa-488 conjugated secondary antibodies (ThermoFisher Scientific) for 1 h at room temperature. Cells were then mounted with proLong™ gold antifade mountant with DAPI (Invitrogen). Immunofluorescence images were captured by NIKON A1R microscopy.

Xu D., Tong X., Sun L., Li H., Jones R.D., Liao J, & Yang G.Y. (2019). Inhibition of mutant Kras and p53 driven pancreatic carcinogenesis by atorvastatin: Mainly via targeting of the farnesylated DNAJA1 in chaperoning mutant p53. Molecular carcinogenesis, 58(11), 2052-2064.

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Evaluating p53 and DNAJA1 Expression

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AsPC-1 cells were plated into a 2-well chamber slide (Nunc Lab-Tek II) at the density of 8 × 10⁴ cells/ml. One day later, cells were transfected with p53^R175H plasmid for 24 h. Cells were washed with PBS, fixed by 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at room temperature, and blocked by 3% BSA in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Cells were then incubated overnight with anti-p53 (SC-99) antibody and anti-DNAJA1 antibody, followed by incubation with Alexa 568– and Alexa 488–conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. After mounting with proLong Gold Antifade Mountant with DAPI (Invitrogen), immunofluorescence images were captured by fluorescence microscopy.

Tong X., Xu D., Mishra R.K., Jones R.D., Sun L., Schiltz G.E., Liao J, & Yang G.Y. (2020). Identification of a druggable protein–protein interaction site between mutant p53 and its stabilizing chaperone DNAJA1. The Journal of Biological Chemistry, 296, 100098.

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