Ab109497

Mice (12 – 14 weeks) were anesthetized and transcardially perfused with PBS followed by 10% formalin in PBS. Spinal cord segments (L3 - L5) were dissected and cryoprotected in 30% sucrose in PBS (n = 5 control, n = 5 CRPS 7 day, n = 6 CRPS 7 week). Tissues were frozen in O.C.T., sectioned coronally (40 μm) on a cryostat (Leica Biosystems), and incubated in blocking buffer (5% donkey serum and 0.3% Triton X-100 in PBS) for 1 hour at room temperature. Sections were incubated overnight at 4°C with primary antibodies rat anti-CD11b (Abd Serotec #MCA711G, 1:500), rabbit anti-TSPO (Abcam, ab109497, 1:500), mouse anti-GFAP (Sigma #G3893, 1:1000) and/or rat anti-CD31/PECAM1 (BD Pharmingen, #550274, 1:1000). After washing (1% donkey serum and 0.3% Triton X-100 in PBS), sections were incubated with appropriate AlexaFluor conjugated secondary antibodies for 2 hours at room temperature. Images were acquired using a Keyence BZ-X800 using the sectioning module to remove non-focused light. We acquired 3 – 5 dorsal horn images per mouse with n = 5 – 6 mice/group, as outlined above, in order to evaluate the relative cellular expression of TSPO, CD11b, GFAP and PECAM1 in the spinal cord dorsal horn. Each image was taken using identical settings including exposure, with 0.4 μm-step z-stacks of 19 slices at 20x or 63x objective magnification.