What is the protocol for immunoperoxidase staining of rat brain sections?
Day 1. 1. Wash sections 2X with gentle rocking/agitation at 4C, 5 min each in ice-cold TBS (0.15M NaCl, 50 mM Tris, pH 7.5). Use 6-well plate with net insert for accelerated washing. One well fits at most 10 sections. Do not overflood the well with TBS since floating sections may be caught at rims of the inserts. Be gentle lifting and lowering the inserts while transferring to prevent sections from breaking due to surface tension of the buffer. 2. Permeabilize sections in 0.5% TX-100 in TBS for 40-45 minutes at 4C with gentle rocking/agitation. 3. Wash sections 2X, 5 min each in ice-cold TBS with gentle rocking/agitation. 4. Block sections in ice-cold vehicle (5% normal horse serum, 0.1% TX-100 in TBS) for 45 minutes at 4C with gentle rocking/agitation. 5. Incubate sections in NeuroMab diluted in ice-cold vehicle. Dilution factors for NeuroMabs should be determined empirically, but generally NeuroMab tissue culture supernatants should be used at 1:2-1:20, purified NeuroMab IgG from 100
