Immunofluorescence for tyrosine hydroxylase (TH) and calcitonin gene related peptide (CGRP)

Isoflurane inhalation was used to deeply anesthetize mice and then in vivo cardiac perfusion was performed by first using a PBS buffer (pH = 7.4), followed by a periodate-lysine-2% paraformaldehyde (PLP) fixative. BAT samples were then harvested and fixed for 36 h at 4°C, followed by a 0.1 M PBS (pH 7.6) rinse and immersion for 24 h at 4°C. Samples were then moved to a 30% sucrose solution in 0.1 M phosphate buffer with 0.1% sodium azide solution for two weeks prior to processing.

Using slight variations to our published protocol [41] samples were embedded and cryosectioned at 20 μm (Richard-Allan HM550VP cryostat) and placed onto gelatin and poly-l-lysine subbed glass slides. Liquid IHC blocker was placed around tissue sections, they were covered with a 4% goat serum super block, and then placed in a humidity chamber at room temperature for 1 h. Tissue sections were then incubated at room temperature for 18 h with primary antibodies against Guinea pig CGRP (Fitzgerald Industries International, Cat# 20 R-CP007; RRID:AB_1282813) or chicken TH (EnCor Biotechnology, Inc., Cat #CPCA-TH; RRID:AB_2737416). After incubation with primary antibodies, sections were washed 4 times in a 1% goat serum Triton x100 PBS solution for 10 minutes per wash. Sections were then incubated for 3 h at room temperature with the following secondary antibodies: Alexa Fluor-488 goat anti-Guinea pig and Alexa Fluor-647 goat anti-chicken (Thermo Fisher Scientific, both diluted 1:100), followed by the 4X-wash procedure. Slides were then covered and mounted with ProLong™ Gold Antifade Mount. Images were obtained using a Keyence epi-fluorescence microscope (Keyence BZ-X710).