Alexa fluor 488 conjugated goat anti rabbit igg

Immunofluorescent staining of paraffin sections was performed as described previously (Abler et al., 2011a (link); Mehta et al., 2011 (link)). Antibodies include: rabbit anti-CCND1 (Abcam #ab16663 Cambridge MA), mouse anti-CTNNB1 (BD Transduction labs # 610153, San Jose, CA), rabbit anti-KI67 (Abcam #ab15580), Alexafluor 488-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch #115-547-003, West Grove, PA), Alexafluor 594-conjugated goat anti-mouse IgG (Jackson ImmunoResearch #111-516-045). Tissue sections were also stained with 4′,6-diamidino-2-phenylindole, dilactate (DAPI) to label cell nuclei.

Animals were anesthetized and transcardially perfused with phosphate buffer solution followed by 4% paraformaldehyde in phosphate buffer solution. Perfused brains were post-fixed in 4% paraformaldehyde for 1 day prior to being sectioned coronally at 100µm with a vibratome. Olfactory bulbs were sectioned on the parasagittal plane at 100 µm thickness. A subset of tissue sections was counter-stained for cell nuclei with TOPRO-3 prior to mounting on slides. Immunohistochemistry was performed on tissue sections as described above. Sections were bathed in 10% FBS in 0.5% TritonX-100/PBS blocking solution for one hour at room temperature. Sections were then incubated overnight at 4°C in rabbit polyclonal anti-ERα primary antibody (06–935, 1:1000; Millipore Sigma). Sections were then washed 3 times in 0.05% tritonX-100 in PBS (5 minutes; 20 minutes; 40 minutes) and incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch). Sections were then washed 3 times in 0.05% tritonX-100 in PBS (5 minutes; 20 minutes; 40 minutes) and mounted on slides for fluorescence microscopy. Analyses investigating the expression of ERα and sex differences in the number of arom+ neurons considered the entire contiguous population of arom+ neurons in the ventral MEA.