Alexa fluor 594 goat anti rabbit immunoglobulin g

In order to investigate how NPY neurons in the central amygdala respond to HFD and cold exposure, 28 transgenic mice expressing GFP under the control of the mouse NPY promoter (NPY-GFP mice) were used. The brain tissues from these mice, upon exposure to ultraviolet light, displayed fluorescence emission in all or the majority of known NPY neurons in the brain (30 (link)). For double labeling co-localization experiments, 5 mice from the cold exposure with HFD group were exposed to cold stress as described above for 1 h per day for 7 weeks, and were then sacrificed by cervical dislocation and decapitation subsequent to perfusion. The brains were dissected, placed overnight in 30% sucrose, and cut into coronal sections of 30 µm thickness using a microtome. Upon rinsing in PBS, the slides were incubated with the primary antibody rabbit anti-mouse c-fos (diluted at 1:2,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA). Next, the secondary antibody Alexa Fluor^® 594 goat anti-rabbit immunoglobulin G (A11037; Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) diluted at 1:250 was added. Red c-fos stain in the c-fos immunohistochemistry test and GFP-positive neurons in GFP-NPY transgenic mice were visualized and counted under a Zeiss Axiophot microscope (Imaging Solutions GmbH).

Paraffin‐embedded tissue sections were stained with an anti‐GLP‐1R monoclonal antibody (Mab 3F52)¹⁴ obtained from Novo Nordisk and an anti‐P504S antibody (AN449‐5ME; Biogenex, Fremont, CA, USA). Sections stained for GLP‐1R were subsequently incubated with Alexa Fluor 488 goat anti‐mouse immunoglobulin G (A‐11017; Life Technologies, CA, USA), and sections stained for P504S were incubated with Alexa Fluor 594 goat anti‐rabbit immunoglobulin G (A‐11012; Life Technologies). The sections were counterstained with 4′,6‐diamidino‐2‐phenylindole and then observed by microscopy.

Fibre cross‐sectional area of soleus muscles was measured. Excised soleus muscles were snap frozen in isopentane cooled by liquid nitrogen and stored at −80 °C for subsequent analysis. Muscle cross sections (10 μm thick) were taken from muscle midbelly. Sections were first treated with 1% bovine serum albumin and normal goat and mouse serum as blocking agents. Sections were incubated overnight with a polyclonal anti‐laminin antibody (Sigma, dilution 1:1000) and then with the secondary antibody, Alexa Fluor 594 goat anti‐rabbit immunoglobulin G (Invitrogen, dilution 1:200). The laminin antibody was used to label the fibre perimeter and facilitate semiautomated fibre area quantification.23 Sections were imaged with a microscope (Leica CTR 6500, Buffalo Grove) fit with a fluorescent camera (Leica DFC365 FX) set for 594 emission fluorescence using a 10× objective. Fibre cross‐sectional areas were measured using a custom‐written macro in ImageJ (National Institutes of Health). Filtering criteria were applied to ensure measurement of actual muscle fibres. These criteria rejected regions with areas below 50 μm² and above 5000 μm² to eliminate neurovascular structures and ‘optically fused' fibres, respectively. Excised soleus muscles were hydrolyzed, and hydroxyproline content was calculated using a colorimetric assay.24