Rat anti perforin

Harvested organs were fixed in 4% paraformaldehyde (PFA) for 24 hours or IHC zinc fixative (BD Pharmingen) for anti-perforin stained tissues. Fixed tissues were then sucrose sedimented in 10%, 20% and 30% steps prior to embedding within optimum cutting temperature (O.C.T.) medium (Tissue Tek). Frozen sections of 5–7 μm thicknesses were cut and left to dry at ambient temperature. These tissues were then stained with primary antibodies: Cleaved Caspase 3 (Cell Signaling Technology), CD31 (Novus Biologicals INC), CD45 (Novus Biologicals INC), glial fibrillary acidic protein (GFAP) (Agilent Technologies INC), rabbit-neuronal marker (NeuN) (Cell Signaling Technology), guinea pig-NeuN (EMD Millipore Corporation), rat anti-perforin (Novus Biologicals INC) and anti-ZIKV rat serum ^{22 (link)} after rehydration with PBS (2X for 10 minutes) and permeabilization with PBST (3X for 10 minutes) at RT overnight. Slides were washed and incubated with appropriate secondary prior to a final wash series and incubation with 4′,6-diamidino-2-phenylindole (DAPI) prior to mounting with SlowFade Diamond Antifade (Fisher Scientific Company LLC). Stained tissues were then analyzed by fluorescence microscopy (BX51; Olympus) or confocal microscopy (TCS SP2; Leica).