Ab1566

Paraffin-embedded whole-fat sections from UC and control patients (n = 4) were mounted on slides. The SP staining was detected using an anti-SP rabbit polyclonal antibody (AB1566; Millipore, Darmstadt, Germany) and the EnVision+ System HRP labeled Polymer Anti-Rabbit kit (DAKO, Carpinteria, CA). The staining was performed at the Translational Pathology Core, University of California at Los Angeles, following a standard procedure described in Millipore’s manual for the primary antibody treatment (1:100 Ab dilution, pretreatment with citrate pH 6.0, antigen retrieval).

L4 DRGs ipsilateral to the cancer-bearing limbs were surgically extracted, fixed in 4% paraformaldehyde, and cryostat sectioned at 25 µm thick transverse to the longitudinal axis. The sections were washed in PBS and incubated with blocking solution containing 10% normal goat serum at 25℃ for 2 h. After overnight incubation with 1:500 rabbit anti-substance P (Millipore, AB1566, Billerica, MA) at 4℃, sections were washed in PBS and incubated with AlexaFluor 488 labelled goat anti-rabbit IgG (Invitrogen, Canada) diluted 1:2000 at 25℃ for 2 h and viewed using a fluorescent microscope. For DRGs containing neurobiotin-injected neurons, these were also incubated with NeutrAvidin Texas Red 1:100 (Vector Laboratories) at 25℃ for 2 h to reveal neurobiotin staining via light microscopy.