Ab193853

EV marker and immuno-related proteins in lysates from EVs and EECs were detected through the use of SDS-PAGE. Separated proteins transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) were blocked with Block Ace reagent (DS Pharma Biomedical, Osaka, Japan), and the membranes were incubated with the following antibodies; rabbit polyclonal anti-human CD63 antibody (1:2000, EXOAB-CD63A-1, System Biosciences), rabbit polyclonal anti-human HSP70 antibody (1:2000, EXOAB- HSP70A-1, System Biosciences), rabbit polyclonal anti-bovine CTSC (1:2000, ab182904, abcam, Tokyo, Japan), rabbit polyclonal anti-pocine IL6 (1:2000, ab193853, abcam), or rabbit polyclonal anti-human ACTB (1:5000, ab1801, abcam). The membranes were then incubated with secondary antibody, horseradish peroxidase labeled goat anti-rabbit IgG (1:5000, Vector Laboratories, Burlingame, CA, USA), and immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, USA). Signals were detected using C-DiGit Blot Scanner (LI-COR) and then band density was assessed with Image Studio DiGit software (version 5.2)^{37 (link)}.

Transverse pieces of thymic samples were fixed in 4% buffered paraformaldehyde over 24 h, and embedded in paraffin and sectioned. The sections were stained by hematoxylin and eosin (HE). The sections were treated as described previously with thymuses instead of lymph nodes [12 (link)]. Immunohistochemical localizations of IFN-γ and IL-6 proteins in the thymus were performed by the IFN-γ antibody (Abcam, ab27919, 1:100) and IL-6 antibody (Abcam, ab193853, 1:100), respectively. The negative controls were handled with antiserum-specific isotype in place of IFN-γ or IL-6 antibody. The antibody binding sites were analyzed and scored as described previously [16 (link)].

Uterine tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and then stained with anti-IL-6 (#ab193853, Abcam, Shanghai, China) and anti-TNF-α (#ab6671, Abcam, Shanghai, China).