Anti mouse igm hrp

For immunohistochemistry, infected tissues from IA mice were fixed in 10% formalin overnight and embedded in paraffin, cut to 4 µm, affixed onto an adhesive slide (Trajan Scientific and Medical, Ringwood, Australia) and dried for about 20 min. After deparaffinization and rehydration in xylene and serial dilutions of ethanol, the antigen retrieval was completed in the pressure cooker (Hawkins, Mumbai, India) with 10 mM sodium citrate buffer (pH 6.0). The tissue samples were then permeabilizated by PBS with gelatin (0.2% w/v) and Triton (0.25% v/v). Afterward, the samples were blocked in 5% BSA in permeabilization solution at RT for 1 h and incubated with antibody supernatant (diluted at 1:10) overnight at 4 °C. The next day, after three 10-min washes in PBS, the endogenous peroxidase of the tissue was inactivated by 3% hydrogen peroxide before incubation with anti-mouse IgM-HRP (Abcam, Cambridge, UK) for 1 h at RT. The samples were developed with 3,3’-diaminobenzidine (DAB) solution (Agilent Technologies, Santa Clara, CA, USA) for 5 min and counterstained with haematoxylin for 1 min at RT. Finally, the samples were dehydrated and permanently mounted and observed under the light microscope (Olympus, Tokyo, Japan). Control groups were incubated with blocking buffer or isotype IgM (6B10) but were otherwise treated the same.