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 488 conjugated donkey anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody is a secondary antibody labeled with the fluorescent dye Alexa Fluor 488. It is designed to detect and bind to rabbit primary antibodies in immunoassays and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (2 mentions)
Alexa fluor 488 nhs, by Thermo Fisher Scientific (1 mentions)
Ix83 microscope, by Hamamatsu Photonics (1 mentions)
Anti lamp1, by Cell Signaling Technology (1 mentions)
Alexa fluor 568 conjugated goat anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Anti eea1, by Cell Signaling Technology (1 mentions)
Anti tbk1, by Abcam (1 mentions)
Ab152, by Merck Group (1 mentions)
Antifade mounting media, by Thermo Fisher Scientific (1 mentions)
Ab51253, by Abcam (1 mentions)
Alexa fluor 568 conjugated donkey anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Trueblack, by Biotium (1 mentions)
Tcs sp8, by Leica (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Bx51p polarizing microscope, by Olympus (1 mentions)
Eosin solution, by Fujifilm (1 mentions)
Mayer s hematoxylin solution, by Fujifilm (1 mentions)
Saponin, by Nacalai Tesque (1 mentions)
Goat serum, by Fujifilm (1 mentions)
13 protocols using alexa fluor 488 conjugated donkey anti rabbit igg antibody
1
Immunofluorescence Staining of STING and Related Proteins
2
Immunohistochemical Analysis of α-Synuclein Phosphorylation
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Deparaffinized sections were washed with running tap water, rinsed with distilled water, and washed once with PBST. Antigen retrieval was performed in 1 mM EDTA (pH 8.0) by boiling for four min and then cooled down for 20 min. After washing three times with PBST, the sections were blocked with 2% bovine serum albumin in PBST for 30 min. Then, the sections were incubated overnight with anti-human FtMt antibody (C65-2; 1:500), rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody (1:500; AB152, Merck Millipore), and p-α-syn at S129 (1:100; ab51253, Abcam, Cambridge, UK) at 4°C. Sections were washed three times with PBST and then incubated with the following fluorescent-labeled secondary antibodies for 1 hr at RT: Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (ThermoFisher, Rockford, IL) and Alexa Fluor 568-conjugated donkey anti-mouse IgG antibody (ThermoFisher). After washing three times with 0.1 M phosphate-buffered saline (PBS), sections were incubated with Hoechst 33258 for 15 min at RT. After washing three times with PBS, TrueBlack (Biotium, Fremont, CA) was applied in a 1:40 dilution for 50 sec for quenching lipofuscin autofluorescence. Sections were washed three times in PBS and then coverslipped using antifade mounting media (ThermoFisher). Fluorescent images were taken using the Leica SP8 confocal microscope (Leitz, Wetzlar, Germany).
3
Histological and Immunohistochemical Analysis of Distal Colon
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For histological analysis, 0.5 cm distal colon was fixed in Zinc Formalin (Polyscience Inc.) for 3 hours and then embedded in paraffin blocks. We then prepared 5 μm paraffin cross-sections, which were used for hematoxylin and eosin (H&E) staining (Mayer’s Hematoxylin solution, 1% Eosin Solution, Wako) following standard procedures. The histological images were captured using the BX51-P Polarizing microscope (Olympus) and processed with the Olympus D.P. Controller 2002 software.
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).
4
Immunofluorescence Staining of AQP3 in Skin
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Skin tissue was postfixed in 4% paraformaldehyde. Tissue samples were embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and frozen by liquid nitrogen. The frozen blocks were cut into 10 μm sections mounted on glass slides. The sections were incubated with a rabbit anti-rat AQP3 antibody (1/200). The sections were treated with an Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1/200, Thermo Fisher Scientific, Waltham, MA, USA). The slides were covered and observed under a fluorescence microscope.
5
Dual-Labeled Immunohistochemistry for Kiss1 and AR
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After hybridization with a DIG-labeled antisense RNA probe for Kiss1 (1 μg/mL), sections were incubated with blocking buffer (1% bovine serum albumin (BSA) in 0.1 M Tris-buffered saline (TBS)) for 1 h at 37°C. Next, sections were washed with 0.1 M phosphate-buffered saline (PBS), followed by incubation with rabbit anti-AR antibody (1:250, rabbit monoclonal, Epitomics, Burlingame, CA, USA) in 0.1 M PBS overnight at 4°C. The following day, sections were washed with 0.1 M PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1:500, Thermo Fisher) and AP-conjugated anti-DIG1 antibody (1:1000) in 0.1 M PBS for 2 h at 37°C. After a 5-min wash, DIG labeling was detected using a 2-hydroxy-3-naphtholic acid-2′-phenylanilide phosphate (HNPP) fluorescent detection set (Roche Diagnostics). Fluorescence images were obtained using confocal laser microscopy (LSM710, Carl Zeiss). Immunoreactive cells were manually counted within single images displayed on a monitor. Cells were counted in every second section through the AVPV and every fourth section through the ARC.
6
HDM2 Expression Analysis in Hematopoietic Cells
7
Quantifying Tissue Hypoxia Levels
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Tissue hypoxia levels were assessed by Hypoxyprobe immunofluorescence as described before54 (link)55 (link). Briefly, animals were administered Hypoxyprobe (Hypoxyprobe, Inc.) via intraperitoneal injection (60 mg kg−1 body weight). Thirty minutes after injection, tissues were harvested, fixed overnight in 4% buffered formalin and embedded in paraffin. Tissue sections were deparaffined, rehydrogenated and incubated with anti-Hypoxyprobe (rabbit anti-PAb2627AP, 1:200 dilution Hypoxyprobe, Inc.) overnight at 4 °C. Hypoxia signalling was detected by applying Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1:1,000 dilution, Life Technologies). Quantification of the fluorescent signalling was performed using the Image-Pro Plus software (Media Cybernetics, Bethesda, MD). The density of the fluorescence was measured. The average densities of 20 areas per samples were determined and the s.e.m. is indicated.
8
Immunofluorescence Localization of HRP and Collagen IV
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Some deparaffinized tissue sections were blocked with 5% fish gelatin in PBS for 1 hr and treated with rabbit anti-HRP and goat anti-collagen type IV polyclonal antibodies at 4°C overnight (Fig. 1 n). Then, they were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody and Alexa Fluor 594-conjugated donkey anti-goat IgG antibody (Life Technologies, Eugene, OR, USA) at room temperature for 1 hr. Finally, intracellular nuclei were counterstained with 4,6-diamidino-2-phenylidole (DAPI), and the immunostained sections were mounted on glass slides with Vectashield (Vector) and observed under a confocal laser scanning microscope (FV-1000; Olympus) (Fig. 1 p).
9
Immunofluorescence Analysis of HepG2-IR Cells
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The HepG2-IR cells treated with BBR were fixed with 4% formaldehyde and permeabilized in 0.5% Triton X-100. The fixed HepG2-IR cells were washed with PBS and blocked with 5% bovine serum albumin in PBS for 60 min at room temperature. Fixed HepG2-IR cells were then incubated with the primary antibodies overnight at 4°C, then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1 : 1000; Life Technologies, Waltham, MA, USA) or Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (1 : 200; ab1501115, Abcam, UK) for 1 h at room temperature, followed by DAPI staining (Solarbio Life Sciences, China) in the dark. Images were taken by a fluorescence microscope.
10
Histopathological Analysis of EV-A71 Infection
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Brain samples from the experimental animals were fixed in 10% formalin in PBS, dehydrated in graded ethanol, and embedded in paraffin before obtaining 4-µm sections for further experiments, including hematoxylin and eosin staining, immunofluorescence assays, and immunohistochemical assays. The EV-A71 antigen was detected using a primary mouse anti-EV-A71 monoclonal antibody (Chemicon, USA) and a secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Sigma, Germany) in immunohistochemical analyses or an Alexa Fluor 594-conjugated donkey anti-mouse IgG antibody (Life Technologies, USA) in immunofluorescence assays. Astrocytes were detected using a rabbit anti-GFAP antibody (Abcam Ltd., UK) as the primary antibody and an Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (Life Technologies, USA) as the secondary antibody. The staining procedure was performed according to a standard protocol [15 (link), 18 (link)]. The histopathological and immunohistochemical analyses were performed using a light microscope (Nikon DS-Ril/Eclipse), and the immunofluorescence assay was performed with a Leica SP8 laser scanning confocal microscope system.
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