Alexa488 donkey anti guinea pig

To explore whether neurons in the NTS projecting to the ACC were activated by painful CP, double immunostaining of FG and FOS was performed in the NTS of CP rats 1 week after FG injection into the ACC. TNBS was administered 1 week before FG injection to establish the CP model. The preparation of brain sections was as previously described. After confirmation of the injection site in the ACC and virus-labeled cell bodies in the NTS, immunostaining was performed using the following antibodies and procedures: after blocking, the ACC sections were incubated in mouse anti-FOS (1:500; ab11959, Abcam) and guinea pig anti-FG (1:200, NM-101, ProtosBiotech, New York, NY, USA) overnight at 4 °C. The sections were then incubated with Alexa594-donkey anti-mouse (1:500; A21203, Invitrogen) or Alexa488-donkey anti-guinea pig (1:500; 706-545-148, Jackson Immunoresearch, West Grove, PA, USA) for 4 h at 4 °C in darkness. The expression of FOS in FG-labeled neurons in the NTS was observed under the confocal microscope.