Immunofluorescence Staining of Rat Brain Sections

The protocol for collection and staining of rat brain sections and cultured cells has previously been described (Greenwood et al., 2016b (link)). The fluorescent images were captured using a Leica DMRB microscope with Leica DFC340FX camera using LAS software. Confocal images were obtained using a Leica SP5-II confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope using LAS software. Primary antibodies were as follows; goat polyclonal anti-Creb3l1 (1:500), mouse anti-cAMP antibody (1:1000; Abcam, ab24851), rabbit polyclonal anti-Nr4a1 (1:50; Santa Cruz, sc-7978), rabbit polyclonal anti-c-Fos (1:25,000; Millipore, PC38) and rabbit anti-HA tag antibody (1:10,000). We have previously described the specificity of the Creb3l1 for Western blotting and immunofluorescent staining applications (Greenwood et al., 2014 (link), 2015a (link), 2016b (link)).