Background

Elucidating the protein organization in neurons and glia is essential for understanding hypothalamic function. Immunofluorescence is a powerful tool to investigate changes in the expression of neuronal and glial proteins and to map activated brain areas at the cellular level. This imaging technique enables the visualization of highly specific protein (antigen) targets using a combination of primary and secondary antibodies. Antigenic determinants of interest in brain tissue are first treated with a monoclonal or polyclonal primary antibody, targeted toward peptides from the species/protein of interest generated in a host (mouse-, rabbit-, chicken-, rat-, donkey-derived, etc., are available), followed by a secondary antibody directed toward the host of the primary antibody and conjugated to a synthetic or natural fluorophore. The antibody targets often labeled in brain tissue sections are glial fibrillary acidic protein (GFAP), neuronal and axonal neurofilaments, neuropeptides, microtubule-associated proteins, blood-brain barrier proteins, pre- and post-synaptic proteins, myelin, markers of inflammation, and numerous antigens associated with specific diseases. Among the useful fluorescent markers for the visualization of antigens in brain tissue are rhodamine, fluorescein, the Alexa Fluor series, and the cyanine dyes. Counterstaining for nuclei using a variety of popular DNA-binding dyes (such as TO-PRO-3 iodide) follows treatment with the secondary antibody.

This method has been previously used by our laboratory to identify neuronal subpopulations in various hypothalamic nuclei involved in energy homeostasis (Dalvi et al., 2017). In particular, using double immunofluorescence staining, we identified GFAP in the hypothalamus and tumor necrosis factor (TNF)-α, a marker of inflammation, in the hypothalamic arcuate nucleus following acute lipid infusion and chronic high-fat diet feeding in mice, which may contribute to a significant alteration in content or expression of appetite-regulating neuropeptides. To the best of our knowledge, this was the first description of TNF-α induction demonstrated by immunofluorescence in the mouse hypothalamic arcuate nucleus after exposure to a high-fat diet. This method can be applied to the identification of other markers of inflammation and endoplasmic reticulum (ER) stress in the hypothalamic regions provided that reliable antibodies are available for detection. This protocol details a generalized procedure for staining frozen brain cryosections ranging from 20 to 30 µm in thickness. Figures 1 and 2 presented in this protocol are confocal images that reveal the expression of GFAP and TNF-α in the hypothalamic arcuate nucleus in 25 µM brain sections of mice treated acutely with lipid or chronically with a high-fat diet (Dalvi et al., 2017).

Figure 1. Effect of acute intralipid treatment at 24 h post-infusion in C57BL/6 mice on hypothalamic arcuate nucleus (ARC) reactive astrocytosis and expression of TNF-α, a marker of inflammation. A-B.Representative images showing immunofluorescence for the expression of GFAP (green) and TNF-α (red) in the hypothalamic ARC of C57BL6 mice at 24 h following acute infusion with saline (A) or Intralipid (B). C. Quantification of the immunofluorescence intensity for GFAP and TNF-α expression in the ARC of C57BL/6 mice at 24 h following acute infusion with saline or Intralipid. Data in the bar graph are expressed as the mean ± SEM (n = 5-6 animals/group); *P < 0.05 versus control. Images in A and B and the graph in C are adapted and modified from Dalvi et al. (2017).

Figure 2. Effect of high-fat diet (HFD, 60% kcal) feeding for 8 weeks in CD-1 mice on reactive astrocytosis and expression of TNF-α in the hypothalamic arcuate nucleus (ARC). A-B. Representative images showing immunofluorescence for the expression of GFAP (green) and TNF-α (red) in the hypothalamic ARC of CD-1 mice fed chow (A) or an HFD (B) for 8 weeks. C. Quantification of immunofluorescence intensity of GFAP and TNF-α expression in the ARC of CD-1 mice fed either chow or an HFD for 8 weeks. Data in the bar graph are expressed as the mean ± SEM (n = 5-6 animals/group); *P < 0.05 versus control. Images in A and B and the graph in C are adapted and modified from Dalvi et al. (2017).