Rat anti mouse cd3

The middle section of the colon tissue was fixed in 10% formalin overnight and 5 μm paraffin embedded cross sections were prepared. After dewaxing and rehydration, tissue sections were boiled for 20 min in 10 mmol/L citrate buffer (pH 6.0). The sections were blocked with blocking buffer for one hour and then incubated with anti-mouse CD68 mAb (for macrophages, 1:100, Abcam Inc, Cambridge, MA, USA, ab955) or rat anti-mouse CD3 (for T cells, 1:50, Abcam Inc, Cambridge, MA, USA, ab11089) mAb separately at 4 °C overnight. After washing, the sections were incubated with Alexa Fluor^® 555 conjugated goat anti-mouse IgG (H+L) (for macrophages, 1:1000, Thermo Fisher Scientific Inc, Waltham, MA, USA, A32727) or Alexa Fluor^® 488 conjugated goat anti-rat IgG (H+L) (for T cells, 1:1000, Thermo Fisher Scientific Inc, Waltham, MA, USA, A-11006) for two hours at room temperature. The sections were then counterstained and mounted with ProLong^® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific Inc, Waltham, MA, USA, P36931). Rabbit isotype control IgG (Abcam Inc, Cambridge, MA, USA, ab27478) at the same dilution was used as control. For each slide, 10 randomly chosen fields (20X) were examined and counted for fluorescent cells. The result for each slide was expressed as the average cell counts in ten fields.