Hrp conjugated goat anti mouse igm

Antibody titres were determined by virion‐based ELISA as previously described.¹⁸, ¹⁹ Pooled heat‐inactivated mouse sera of respective groups were tested at 1:100 dilution for IgM, and 1:500 dilution for IgG. For IgG subtyping studies, pooled sera were serially diluted from 1:250 to 1:32 000 for IgG3, 1:512 000 for IgG1 and IgG2b, and 1:8 192 000 for IgG2c. ZIKV‐specific IgG subtypes are expressed as antibody titre, defined as the greatest reacting dilution before the OD value reaches baseline control (pooled 0 dpi sera). HRP‐conjugated goat anti‐mouse IgM (Santa Cruz Biotechnology, Dallas, TX, USA), IgG (Merck, Kenilworth, NJ, USA), IgG1 (Santa Cruz Biotechnology), IgG2b (Santa Cruz Biotechnology), IgG2c (Southern Biotech, Birmingham, AL, USA) and IgG3 (Santa Cruz Biotechnology) antibodies were used. IgG2c was tested in lieu of IgG2a since only IgG2c gene is present in C57BL/6 mice.⁴⁷, ⁶⁷ ELISAs were developed using TMB substrate and terminated with stop reagents (Sigma‐Aldrich). The absorbance was measured at 450 nm. ELISA readings were conducted in duplicates.

Mouse brain cortex was homogenized with M-PER (Thermo Scientific) containing 1× Halt protease & phosphatase inhibitor cocktail (Thermo Scientific). The brain lysate (3 µg/lane) was loaded on SDS-PAGE for western blot (WB) analysis. After blocking the membrane with 5% skim milk (Wako Pure Chemical Industries, Japan) in 0.1% Tween-TBS for 30 min at room temperature, the anti-mouse HSC70 antibody (1:4000, Abcam) and anti-rabbit β-actin antibody (1:1000, Cell Signaling Technology) were used as the primary antibodies, and HRP-conjugated goat anti-mouse IgM (1:2000, Santa Cruz) and HRP-conjugated goat anti-rabbit IgG (1:2000, Santa Cruz) were used as the secondary antibodies. Amersham™ ECL™ Western Blotting Detection Reagents (Sigma-Aldrich) was used for the detection of the bands according to the manufacture’s protocol.