Immunofluorescence Assay for HMGB1, KSHV Proteins

Immunofluorescence assay (IFA) was performed as previously described (Lee M. S. et al., 2014 (link); Kang et al., 2021a (link)). The primary antibodies used were anti-HMGB-1 (Abcam), anti-KSHV ORF50 (Bioss), anti-KSHV K8.1 (Santa Cruz), and anti-LNA (Abcam) antibodies. The secondary antibodies used were Alexa Fluor 568-conjugated anti-mouse IgG (Thermo Fisher Scientific) and Alexa Fluor 568-conjugated anti-rabbit IgG (Thermo Fisher Scientific) antibodies. A concentration of 500 ng/mL of 49,6-diamidino-2-phenylindole (DAPI) was used to stain nucleic acids. The stained samples were observed under a Nikon Eclipse E400 microscope (Nikon, Tokyo, Japan) under the same conditions.

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Lee M.J., Park J., Choi S., Yoo S.M., Park C., Kim H.S, & Lee M.S. (2023). HMGB1, a potential regulator of tumor microenvironment in KSHV-infected endothelial cells. Frontiers in Microbiology, 14, 1202993.

Publication 2023

anti-HMGB-1 anti-KSHV ORF50 Alexa Fluor 568-conjugated anti-mouse IgG Alexa Fluor 568-conjugated anti-rabbit IgG Nikon Eclipse E400 microscope

Alexa 568 Anti igg Antibodies Hmgb Immunofluorescence assay Kshv Microscope Mouse Nucleic acids Rabbit

Corresponding Organization : Inha University

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Variable analysis

independent variables

Primary antibodies used: anti-HMGB-1, anti-KSHV ORF50, anti-KSHV K8.1, and anti-LNA antibodies
Secondary antibodies used: Alexa Fluor 568-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG antibodies

dependent variables

Immunofluorescence signal observed under a Nikon Eclipse E400 microscope

control variables

Staining conditions, including a concentration of 500 ng/mL of DAPI to stain nucleic acids
Microscope settings and conditions used to observe the stained samples

controls

No positive or negative controls were explicitly mentioned in the provided information.

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