Rabbit anti mouse igm

Immulon 4HBX 96-well plates (Fisher, 14–245-153) were coated with 50 μL protein antigens at 2μg/ml in PBS and incubated overnight at 4°C. Plates were washed three times and blocked for one hour with 125 μL of blocking buffer (PBS with 3% goat serum, 0.5% nonfat milk, 0.1% Tween 20). Two-fold serial dilutions of serum samples were made in blocking buffer starting at a 1:50 dilution. Plates were washed three times, then diluted serum was added to plates and incubated for two hours. Detection antibodies conjugated to HRP were diluted in blocking buffer: rabbit anti-mouse IgM (Jackson Immunoresearch, 315–035-049) diluted 1:5000; goat anti-mouse IgK (Novus, NB7549) diluted 1:5,000; goat anti-mouse IgG1 (Southern Biotech, 1071–05) diluted 1:5,000; goat anti-mouse IgG2a (Southern Biotech, 1081–05) diluted 1:2,500. Plates were washed three times and diluted detection antibodies were added to plates and incubated for one hour. Plates were washed three times and 50μL of room temperature KPL SureBlue TMB substrate was added to wells. Plates incubated for 5 minutes before quenching with 25μL of 250mM HCl. Well absorbance was read at 450 nm. All wash steps were conducted using PBS with 0.1% Tween 20.

BALB/c tissues (brain and skeletal muscle) were dissociated by Polytron homogenizer (4,000 rpm) in homogenizing buffer (Tris-HCl 0.5 M, pH 6.8, SDS 10%, and Mili-Q water) as described by Haury et al. (1994) (link). Tissue extracts were fractioned by electrophoresis in 10% polyacrylamide gel under denaturing conditions at 50 mA until 6 cm of migration. Proteins were transferred from the gel to a nitrocellulose membrane by a semi-dry electrotransfer (Semi-Dry Electroblotter B) for 60 min at 0.8 mA/cm². After transfer, the membrane was kept in 50 ml PBS/Tween 20 (Bio-Rad) at 0.2% vol/vol shaking for 18 h at room temperature.
Incubation of the membrane with cell culture supernatants or serum samples was performed in a Miniblot System Cassette (Immunetics), which allows the simultaneous incubation of 28 different samples in separated channels. Supernatants were diluted 1:2, and serum samples were diluted 1:100. After wash, membranes were incubated with secondary antibodies conjugated to alkaline phosphatase anti-IgM or anti-IgG diluted 1:2,000 (rabbit anti-mouse IgM; Jackson ImmunoResearch; goat anti-mouse IgG; Southern Biotech). Substrate NBT/BCIP (Promega) was added after wash. Reaction was developed shaking at room temperature and stopped with Milli-Q water. Colloidal gold staining was performed after scanning membranes.