Anti er tr7

Fluorescein-, phycoerythrin-, or biotin- labeled anti-B220 (clone:RA3-6B2), anti-CD3 (Clone:145-2c11), anti-Thy1.2 (clone:30-H12), anti-CD11c (clone:N418), anti-CD21/35 (clone:7G6), anti-FDC-M1 (clone:FDC-M2) were obtained from BD Pharmingen. Anti-ER-TR7 was from Abcam plc., anti-PCAM-1 (clone:390), and anti-PNAd (clone:MECA-79) were from Biolegend. Anti-IgG1 and goat anti-hamster IgG and fluorescein- or phycoerythrin-labeled streptavidin were all purchased from BD Bioscience. NP-OVA and NIP-BSA were purchased from Bioresearch Technology. Lymphotoxin α1β2 was purchased from R&D System. CXCL13 (cat no.:300-47), CCL19 (cat. No:300-29B), CCL21 (cat. No:300-35), CXCL12 (cat no:300-28A), and sRANKL (cat. No:310-01) were purchased from Peprotech. Medgel was obtained from MeDGEL Co., LTD (Japan).

For analysis of thymic medulla and cortex by immunohistology, thymi from GCV treated TK⁻ and TK⁺ mice were fixed in 4% formalin and embedded in paraffin blocks. Sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by light microscopy. For immunofluorescence, serial sections (5 μm) from OCT-embedded frozen tissues or primary cultured cells were fixed in cold acetone or 4% polyoxymethylene and blocked in PBS/1% BSA, washed in PBS/0.05% Tween and incubated with optimal dilutions of fluorochrome-conjugated antibodies: Alexa Fluor® 488 anti-I-A/I-E, anti-CD31-PE (Biolegend), anti-CD11c-PE, anti-CD11b-FTIC (BD Pharmingen), and anti-F4/80-PE (eBioscience), or with first Abs: anti-cytokeratin 5, anti-FSP1, anti-ER-TR7 (Abcam), anti-cytokeratin 8 (Tromal-1; Developmental Studies Hybridoma Bank), Biotinylated UEA-1, anti-vimentin (BD Pharmingen), anti-α-SMA, anti-Pan-CK (Sigma, Cat no. C5992), anti-CD140a/PDGFRα (R&D Systems) and anti-MTS15 Ab for 2 h at room temperature before washing and incubating with secondary reagents: Alexa Fluor® 546 Goat anti-Rabbit/mouse IgG (H+L), Alexa Fluor® 488 Goat anti-Rat IgG (H+L) (Invitrogen), Dylight 488 Goat anti-Rabbit/mouse IgG (ZSGB-Bio) and streptavidin-PE (BD PharMingen). Control slides were incubated with isotype-matched Ig. Images were acquired with a two-photon microscopy (Carl Zeiss, Inc.).

A histological study was performed as previously described [22 (link)]. For immunohistochemistry, blocking of endogenous peroxidase activity was performed using 0.03% H₂O₂ in methanol. Obtained liver sections were treated with the anti-ER-TR7 (Abcam, Cambridge, MA, USA) antibodies overnight at 4 °C. After treatment with secondary antibodies, the substrate reaction was performed using 3,3′-diaminobenzidine (Dojindo, Kumamoto, Japan) solution.
According to Kleiner et al. [30 (link)], the NAFLD activity score (NAS) was calculated. Quantitative five grades assessment of Oil Red O staining was carried out by scoring of positive areas. Quantitative estimations of ER-TR7 and Sirius-red positive areas were carried out of the positive areas in five fields. Briefly, for each animal, bright field images of stained sections were captured around the central veins at 400-fold magnification using a digital camera (DP72, Olympus, Tokyo, Japan), and quantitatively estimated using WinROOF image processing software (Mitani, Tokyo, Japan). The results were shown as the mean of five different fields in each section.