Transfection and immune staining were performed in Millicell EZ chamber slides (Millipore Sigma, Temecula, CA, USA). Cells were fixed by 4% formaldehyde in PBS for 15 min, permeabilized by 0.4% Triton X-100 on ice for 10 min, and stained with rabbit anti-STING antibody (1:400; Novus bio, NBP2-24683) overnight at 4°C, or in the case of cells transfected with FLAG-STINGΔTM, stained with Cy3-conjugated anti-FLAG antibody (Sigma, A9594). For recombinant STING, proteins were conjugated with NHS–Alexa Fluor 488 (Thermo Fisher Scientific). Other primary antibodies used are anti-TBK1 (Abcam, ab235253), anti-LAMP1 (Cell Signaling, 9091S), and anti-EEA1 (Cell Signaling, 3288S). After washing with PBS containing 0.05% Tween 20, cells were stained with secondary antibodies including Alexa Fluor 568–conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific, no. A-11011) and Alexa Fluor 488–conjugated donkey anti-rabbit IgG antibody (Thermo Fisher Scientific, no. A-32790). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and Golgi apparatus was stained with Golgi-ID green detection kit (Enzo Life Sciences, 51028-GG). Cells were imaged with an inverted Olympus IX83 microscope equipped with a Hamamatsu ImagEM high-sensitivity camera at the Swanson Biotechnology Center (MIT).
Alexa fluor 568 conjugated goat anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568–conjugated goat anti-rabbit IgG antibody is a fluorescent-labeled secondary antibody. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 nhs, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Ix83 microscope, by Hamamatsu Photonics (1 mentions)
Anti lamp1, by Cell Signaling Technology (1 mentions)
Anti eea1, by Cell Signaling Technology (1 mentions)
Anti tbk1, by Abcam (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Mebstain apoptosis tunel kit direct, by Medical & Biological Laboratories (1 mentions)
Ab10983, by Abcam (1 mentions)
Bz x710 all in one fluorescence microscope, by Keyence (1 mentions)
Sc 2027, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
A11036, by Thermo Fisher Scientific (1 mentions)
Ab76541, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Alexa fluor 568 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
4 protocols using alexa fluor 568 conjugated goat anti rabbit igg antibody
1
Immunofluorescence Staining of STING and Related Proteins
2
Immunostaining and TUNEL Assay for Cellular Analysis
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For immunostaining, differentiated cells were fixed by cold Acetone/Methanol (1:1). The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human UCP1 antibody (ab10983; Abcam, Cambridge, UK) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor® 568-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc.). To evaluate the viability of spheroids, TUNEL assays were performed. Spheroids were fixed by using 1% paraformaldehyde in a buffered solution and embedded in paraffin blocks. Spheroid slices were subjected to TUNEL assays, which were performed by using MEBSTAIN Apoptosis TUNEL Kit Direct (8445, Medical and Biological Laboratories Co. Ltd., Nagoya, Japan) according to the manufacturer’s instructions.
The samples were observed either under BZ-X710 All-in-One fluorescence microscope (KEYENCE Corp) or a confocal laser scanning microscope FLUOVIEW FV1000 (Olympus Corp., Tokyo, Japan).
The samples were observed either under BZ-X710 All-in-One fluorescence microscope (KEYENCE Corp) or a confocal laser scanning microscope FLUOVIEW FV1000 (Olympus Corp., Tokyo, Japan).
3
Immunofluorescence Staining of Cdx2 in Cells
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Cells were grown for 2 days on a fibronectin-coated plastic-bottom dish (35 mm µ-Dish; ibid.), fixed with 4% paraformaldehyde/phosphate buffer for 1 hour at room temperature, and then washed three times with 0.1% Triton X100/PBS. Cells were blocked with blocking solution (0.5% normal goat serum [Vector] in PBS +0.1% Triton X-100 [PBST]) and incubated overnight at 4°C with primary antibody diluted in blocking solution. Cells were then washed three times with PBST, incubated with Alexa Fluor 568–conjugated goat anti–rabbit IgG antibody (1∶500; A11036, Invitrogen) for 1 hour at room temperature, washed in PBST, counterstained with DAPI (1 µg/ml; Invitrogen), and imaged. The rabbit anti-Cdx2 antibody (ab76541, Abcam) was used at 1∶500 dilution.
4
Multicolor Immunofluorescence Imaging of Oral Epithelial Cells
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For double-immunofluorescence examinations of caspase 3 and CD68, Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen), respectively, were used as the secondary antibodies. For caspase 3 and hemoglobin, the anti-caspase 3 was pre-labeled with Alexa Fluor 568 (ZenonTM kit, Thermo Fischer Scientific), while Alexa Fluor 488-conjugated goat anti-rabbit IgG was used as the secondary antibody for hemoglobin. Labeled sections were covered with Immunoselect Antifading Mounting Medium DAPI (Dianova, Hamburg, Germany). K17 immunofluorescence was performed for confocal microscopy to investigate K17+ keratin filament distribution within oral squamous epithelial cells on tissue sections by an indirect method using Alexa Fluor 568-conjugated goat anti-mouse IgG (H + L) (Invitrogen) as the secondary antibody, as described elsewhere22 (link). The slides were examined with an LSM710 confocal laser scanning microscope (Zeiss, Oberkochen, Germany). Z-stack images were obtained at constant Z intervals of 0.5–1.0 µm. Images and montages were generated using Zeiss Zen software (Zeiss) and Adobe Photoshop (Adobe Systems, San Jose, CA, USA).
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