A 11056

For simultaneous visualization of both tracers, one series of brain tissue (n = 4) underwent fluorescent double-label immunohistochemical processing. Tissue was rinsed from the cryoprotectant storage solution with several washes in KPBS, and then incubated for 72 h at 4 °C in a blocking solution [KPBS containing 0.3 % Triton X-100, 2 % normal donkey serum (017-000-001; Jackson ImmunoRe-search, West Grove, PA, USA)], with both primary antibodies: anti-FG (1:10,000) and anti-CTB (1:5000). After rinses in KPBS, tissue was incubated for 1 h in the dark in the blocking solution containing the secondary antibodies: Alexa 488 anti-rabbit (1:200; A21206; Invitrogen, Carlsbad, CA, USA) and Alexa 546 anti-goat (1:200; A11056; Invitrogen, Carlsbad, CA, USA), both raised in donkey serum. Following rinses in KPBS, tissue was mounted in semidarkness onto slides (SuperFrost Plus), dried, coverslipped with Vectashield HardSet Mounting Medium with DAPI (H-1500; Vector Labs, Burlingame, CA, USA), and stored at 4 °C until analysis.

The recovered control, sham and CCI sciatic nerves were prepared for immunohistochemical examination using mouse anti rat CD68 antibody (MCA341R, AbD Serotech, Raleigh, NC) and Alexa fluor 488 donkey anti-mouse secondary antibody (A-21202, Invitrogen, Carlsbad, CA) to assess the presence of macrophages that infiltrate the nerve. The double immunofluorescence studies were also performed to reveal the expression of COX-2 and PGE2 in relationship to the CD68 positive infiltrating macrophages using, rabbit anti-COX-2, 1:100 (ab15191, AbD Serotech) and rabbit anti-prostaglandin E2 antibody, 1:100 (ab2318, AbD Serotech) along with mouse CD68, 1:100 (MCA341R, AbD Serotech) in treated and untreated rat groups. The secondary antibodies used were Alexa fluor 488 donkey anti-mouse, 1:200 (A-21202, Invitrogen), Alexa fluor 546 donkey anti-goat, 1:200 (A-11056, Invitrogen) and Alexa fluor 647 donkey anti-rabbit, 1:200 (A-31573, Invitrogen). All the antibody dilutions were prepared using 1:20 normal donkey serum in 1× PBS, pH 7.4. Antigen retrieval was performed during double immunofluorescence with COX-2 and CD68 primary antibodies, using sodium citrate buffer (10mM sodium citrate, 0.1% Tween 20, pH 8.5). Antigen retrieval was not required during double immunofluorescence with PGE2 and CD68 primary antibodies.