Fast free of acrylamide clearing tissue (FACT) is a modified sodium dodecyl sulfate-based clearing
protocol for the chemical clearing of lipids that completely preserves fluorescent signals in the
cleared tissues. The FACT protocol was optimized to image translucent immunostained brain and
non-nervous tissues. For this purpose adult male Chukar partridge (Alectoris chukar) was used as a
model. After clearing the tissues, 1 or 2 mm-thickness sections of tissues were immunolabeled. The
paraventricular nucleus in the hypothalamus (2-mm section) was cleared with FACT, and then was
stained with gonadotropin-inhibitory hormone (GnIH) antibody and Hoechst. Simultaneously, immunohistochemical
(IHC) staining of cryosectioned brain (30 μm) was done by GnIH-antibody. The
FACT protocol and staining of cell nuclei of nine other tissues were done by a z-stack motorized
fluorescent microscope. GnIH-immunoreactive neurons were found by FACT and IHC during the
breeding season in male partridges. Deep imaging of the kidney, duodenum, jejunum, lung, pancreas,
esophagus, skeletal muscle, trachea, and testis were also done. The FACT protocol can be used for
the three-dimensional imaging of various tissues and immunostained evaluation of protein markers