Anti hla dp e 20

Anti–HLA-DP (HL-38, Sigma-Aldrich), anti–HLA-DP-DQ-DR (WR18, Bio-Rad), anti-rat RT1B (OX-6, BD Biosciences), anti–MHC class II (M5/114.15.2, eBioscience), anti-TSHR (2C11, Santa Cruz Biotechnology), anti-His (9F2, Wako), and anti-Flag (purified IgG, M2, Sigma-Aldrich) Abs were used for flow cytometry. anti-Flag (biotin-conjugated, M2, Sigma-Aldrich) and anti-His (28-75, Wako) Abs were used for immunoprecipitation. anti-Flag (purified IgG, M2, Sigma-Aldrich) and anti-His (9F2, Wako) Abs were used for Western blotting. Anti–HLA-DP (E-20, Santa Cruz Biotechnology) and anti-TSHR (HPA026680, Sigma-Aldrich) polyclonal Abs were used for immunofluorescence staining and PLA. To produce human anti-TSHR mAbs, the V regions of the mAbs were synthesized according to the published sequence (accession numbers: M22 heavy chain, 3G04_B; λ chain, 3G04_A; K1-18 heavy chain, AMF37397; λ chain, AMF37401; K1-70 heavy chain, 2XWT_A; and λ chain, 2XWT_B). For germlined K1-18, some mutants were generated using the QuikChange Multi Mutagenesis Kit (Agilent Technologies) from wild-type K1-18 V regions (heavy chain, N31S, Y54G, and V97A; λ chain, N31S and N32S). These V regions were cloned into pME18S expression vectors containing the secreted form of IgG1 constant region as previously described (14 (link)). HEK293T cells were purchased from RIKEN Cell Bank.

Paraffin-embedded tissue sections from GD patients (CS802478 and CS809728) and individuals without GD (CS804207 and CS811532) were purchased from OriGene. These tissue sections were stained with anti–HLA-DP (E-20, Santa Cruz Biotechnology) and anti-TSHR (HPA026680, Sigma-Aldrich) polyclonal Abs, followed by Alexa Fluor 647–conjugated anti-goat IgG or Alexa Fluor 488–conjugated anti-rabbit IgG Abs (Molecular Probes). The stained tissue sections were analyzed using an Axioplan 2 fluorescence microscope (Zeiss).

A Duolink in situ PLA kit was used according to the manufacturer’s instructions (Sigma-Aldrich) for in situ PLAs. Paraffin-embedded tissue sections from the same patients as used in immunofluorescence stain were incubated with anti–HLA-DP (E-20, Santa Cruz Biotechnology) and anti-TSHR (HPA026680, Sigma-Aldrich) polyclonal Abs, and red fluorescent protein PLA signals were developed using anti-goat MINUS and anti-rabbit PLUS PLA probes. Nuclei were stained with 4′,6-diamidino-2-phenylindole fluorescence dye. The assayed tissue sections were analyzed with an Olympus IX83 inverted microscope. The numbers of fluorescent spots from PLA signals per follicle were quantified using ImageJ software.