Superfrost plus

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.

Immediately following slicing, sections were 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 ImmunoResearch, West Grove, PA, USA)], with the primary antibody: anti-c-Fos antibody raised in rabbit (1:10,000; Millipore, Billerica, MA). After rinses in KPBS, tissue was incubated for 1 h in the dark in the blocking solution containing the secondary antibody: anti-rabbit Alexa 488 (1:200; A21206; Invitrogen, Carlsbad, CA, USA). Following rinses in KPBS, in semidarkness tissue was mounted 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.