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Cy3 conjugated goat anti rabbit igg h l

Manufactured by Abcam
Sourced in United Kingdom

Cy3-conjugated Goat Anti-rabbit IgG (H+L) is a secondary antibody used for immunodetection. It binds to the heavy and light chains of rabbit immunoglobulin G (IgG) and is conjugated to the fluorescent dye Cy3.

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3 protocols using cy3 conjugated goat anti rabbit igg h l

1

Tracking Nanoparticle Biodistribution in Gastric Cancer

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DiR (Bridgen, Beijing, People’s Republic of China) was incubated with hCTL membrane-derived vesicles for 30 min to monitor the biodistribution of the nanoparticles in vivo. A subcutaneous xenograft model of gastric cancer was established by injecting 106 MKN-45 cells in 100 μL of PBS subcutaneously in male Balb/c nude mice. Treatment started when the tumor volume reached ~300 mm3. Three tumor-bearing Balb/c nude mice received LDI (2 Gy), and three did not. They were all injected intravenously with TPNPs the day after LDI. Then, the mice were anesthetized and scanned at different time intervals using a Maestro™ Automated In Vivo Imaging System. For resected organ imaging, the animals were euthanized, and the tumors and organs were excised and imaged.
DiO-loaded TPNPs were injected into the tail vein of tumor-bearing mice. After 24 h, the mice were sacrificed under deep anesthesia. Tumors were harvested and processed for immunostaining. Frozen sections were stained using rabbit anti-human CD31 (Abcam, Cambridge, UK) followed by secondary antibodies with Cy3-conjugated Goat Anti-rabbit IgG (H+L; Abcam). Tumor cells were stained using DAPI. Images were taken using a confocal microscope.
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2

Quantifying HIF-1α Expression After Irradiation

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To measure HIF-1α expression, cells were plated in two separate 8-well chamber slides, a negative control slide and a 10 Gy irradiated slide. Thirty minutes after irradiation, the slides were washed three times with cold PBS and subsequently fixed in a 10 min cold acetone bath. Cells were then stained with rabbit anti-HIF-1α (cat. no. ab2185; Abcam, Cambridge, UK), dilution 1:500 and Cy3 conjugated goat anti-rabbit IgG (H+L; cat. no. 111-167-003 Jackson Immuno Research Laboratories Inc., West Grove, PA) 1:400 was the secondary antibody (red). Hoechst 33342 (3.75 mg/ml; Sigma-Aldrich®, St. Louis, MO) dilution 1:3,000 was used for nuclear staining (blue). Images were quantified using the image-procesing program FIJI (ImageJ) (38 (link)) to integrate HIF-1α intensity normalized to the number of nuclei in each field of view. The color channels in RGB images were split to allow for the application of intensity thresholds, and an intensity threshold also removed artifacts from the images. In all images, there were no counts observed in the green channel. Objects on the edges of images were excluded from quantification, and only objects 25 pixels in size or larger were considered to eliminate extracellular speckle.
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3

Immunofluorescent Analysis of Apoptosis Markers

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Immunostaining analysis was performed by immunofluorescent tagging for Bcl2, cleaved caspase-3 and phospho-β-catenin in the tissue sections following our existing protocol. Briefly, the frozen sections were fixed with ice-cold acetone. Sections were then washed 3 times in PBS, permeabilized with 0.3% Triton X-100 in PBS, and blocked with 5% bovine serum albumin in PBS with Tween 20. Immunostaining was performed with rabbit anti-rat caspase-3 (1:100), rabbit anti-rat Bcl2 (1:100), and rabbit anti-rat phosphoβ-catenin (1:100) overnight at 4°C. The sections were washed 3 times in PBS and incubated for 1 h at room temperature in the dark with Cy3-conjugated goat anti-rabbit IgG (H&L) (Abcam, Cambridge, UK) in PBS, and the cell nuclei were counterstained with 0.5 µg/ml DAPI (Beyotime, Shanghai, China) for 2 min. The sections were covered with mounting medium, and the coverslips were sealed with nail polish to prevent drying and fluorescence quenching. The samples were observed and photographed under a fluorescence microscope (Leica, Wetzla, Germany).
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