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EdU (Click-iT EdU Imaging Kit; Invitrogen) was injected intraperitoneally from P3 to P5 once a day at a dose of 50 ?g/g. DT (List Biological Laboratories) was injected intraperitoneally at 12.5 ng/g, 25 ng/g, or 37.5 ng/g at P2
placed in RPMI1640 with 10% FBS, 50?U/mL penicillin and streptomycin.17 The cells were stored overnight. The floating containers were washed with PBS, and cells underwent incubation in freshly prepared medium overnight. Then, adherent macrophages underwent further incubation in serum-free RPMI1640 at 37°C for 2?h and subsequent treatment with LPS (List Biological Laboratories, Escherichia coli 055:B5), Olaparib, or their combination
One day after transfer, recipient mice were immunized with MOG35-55 peptide in CFA and then given an intraperitoneal injection of pertussis toxin (PTX, 500?ng/injection) (List Biological Laboratories, Campbell, CA, USA)12
water at 2 mg/ml, mixed at a 1:1 ratio with complete Freunds adjuvant (CFA, Sigma-Aldrich Co., St. Louis, MO, USA), supplemented with 4 mg/ml of Mycobacterium tuberculosis. Pertussis toxin (200 ng List Labs, USA) was dissolved in 100 ?l of phosphate-buffered saline (PBS) and injected i.p. the same day of the immunization and 48 h later32. Mice were scored daily for EAE symptoms starting at 6 d
For immunofluorescence staining, tissue sections were stained with camelid anti-Stx1 and Stx2 (List Biological Laboratories, Campbell, CA, USA) followed by goat anti-llama:FITC (Invitrogen). For Stx2 ELISA and mass spectrometry, proteins were extracted and processed from paraffin-embedded kidney sections as previously described . Stx2
Immunization and challenge. Mice were immunized weekly for 6 weeks by oral gavage 167 with 5mg ground blanched peanut (Western Mixers Produce & Nuts) and 10µg cholera 168 toxin (CT) (List Biologicals) in 200µl of 0.2M sodium bicarbonate buffer per mouse