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Dylight 488 conjugated donkey anti goat igg

Manufactured by Abcam
Sourced in Germany

DyLight 488-conjugated donkey anti-goat IgG is a secondary antibody that binds to goat immunoglobulin G (IgG) and is conjugated to the DyLight 488 fluorescent dye. This product is commonly used in immunofluorescence and Western blotting applications to detect and visualize goat primary antibodies.

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2 protocols using dylight 488 conjugated donkey anti goat igg

1

Immunofluorescence Labeling of Cell Samples

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Samples were fixed in paraformaldehyde (4% w/v in 1× PBS) for 15 min. Samples intended for labeling of secreted proteins (namely ALB and OPN) were treated with brefeldin A (10 µg/ml, R&D Systems, 1231/5) for 2 hr prior to fixation. Fixed samples were permeabilized with Triton X-100 (0.25% v/v in 1× PBS) for 10 min and incubated in blocking buffer (5% v/v donkey serum and 0.1% v/v Triton X-100 in 1× PBS) for 1 hr at room temperature. We incubated samples for 1 hr at room temperature or overnight at 4°C with one or two of the primary antibodies listed in Table 2 diluted in blocking buffer. The next day, we incubated samples for 1 hr at room temperature with one or two of the following secondary antibodies diluted in blocking buffer: DyLight 488-conjugated donkey anti-rabbit IgG (1/50 from stock, Abcam, ab96919), DyLight 550-conjugated donkey anti-mouse IgG (1/50 from stock, Abcam, ab98767), and DyLight 488-conjugated donkey anti-goat IgG (1/50 from stock, Abcam, ab96935). Samples were mounted in Fluoromount G with DAPI (Southern Biotech, 0100–20) and imaged no earlier than the day after mounting using an Axiovert 200M microscope (Carl Zeiss, Inc.) and associated Zen Pro software. In order to capture entire arrays as one image for later analyses, we utilized the tiling feature of Zen Pro.
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2

Spatial Localization of Autophagy Markers

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Spatial localisation of p62, ubiquitin and LC3B was investigated by triple immunofluorescence staining and subsequent 3D reconstruction. One‐μm‐thick FFPE tissue sections (HCC) were cut, dewaxed, rehydrated and pre‐treated with Target Retrieval Solution (Agilent Technologies, Ratingen, Germany) at pH 9.0 for 20 min at 97 °C. For triple labelling immunofluorescence the primary antibodies anti‐p62 (MBL, Nagoya, Japan), anti‐ubiquitin (Santa Cruz Biotechnology) and anti‐LC3B (Nano Tools, Hamburg, Germany) were used. The secondary antibodies used were Cy5‐conjugated donkey anti‐rabbit IgG (Dianova, Hamburg, Germany), DyLight® 488‐conjugated donkey anti‐goat IgG (Abcam) and Alexa Fluor 555‐conjugated donkey anti‐mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA). Anti‐p62 antibody was labelled with Cy5, anti‐ubiquitin antibody with DyLight® 488 and anti‐LC3B antibody with Alexa Fluor 555; all were diluted 1:100 and were incubated for 1 h at RT (see supplementary material, Table S3). DNA was stained with DAPI; image analysis and 3D reconstruction were performed as described above.
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