Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

ANA detection

US Upneet K. Sokhi
ML Mark P. Liber
LF Laura Frye
SP Sungho Park
KK Kyuho Kang
TP Tania Pannellini
BZ Baohong Zhao
RN Rada Norinsky
LI Lionel B. Ivashkiv
SG Shiaoching Gong
request Request a Protocol
ask Ask a question
Favorite

Blood was collected from 6- to 7-month-old female mice from all groups except the Tnfaip3−/− group (3–4 weeks) and allowed to clot for 1 h at room temperature. Serum was prepared by centrifugation at 15 871 × g for 2 min. Supernatant was saved, aliquoted, and immediately stored at −80 °C prior to analysis. To detect ANA levels, samples were loaded onto a Hep-2 antigen substrate slide (MBL, Cat. # AN-1016) and incubated with a 1:800 dilution of Alex Fluor® 488 goat anti-mouse IgG (Jackson ImmunoResearch, Cat. # 115-546-003). Mounting medium (Fluoromount-G, Southern Biotechology, Cat. # 0100-01) plus Prolong Gold DAPI (Life Technologies, P-36931) was added, slides were sealed, and visualized with a Nikon Eclipse NI-E Fluorescence microscope (Nikon Instruments, Melville, NY, USA). Green fluorescence corresponds to IgG antibody location.

Copyright and License information: The Author(s) ©2018
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A