Expression of molecular markers in mouse and human stellate ganglia
Daniele Neri, Lori Zeltser, Seoeun Lee, Alexandre J Lafond
Abstract
Protocol for immunohistochemistry
Before start
The human stellate ganglia (SG) were provided by Dr. Olujimi Ajijola at UCLA. The tissue was collected post-mortem from a 44-year-old female donor and dissected immediately after the donor's death.
The dissection procedure of murine SG is described in the "Tissue Harvesting" section of our protocol, "Injection of cholera toxin subunit B in the interscapular brown adipose fat and Stellate Ganglion dissection."
Steps
Postfixation of the tissue
Both mouse and human stellate ganglia (SG) were fixed in 4% PFA in 0.1M PB overnight at 4°C, washed with Phosphate Buffer Saline (PBS) 4°C for at least 1 h
Encasing and slicing
Ganglia were encased in Optimal Cutting Temperature compound (Tissue-Tek) cut into 10 μm cryo-sections on slides, and stored at -80°C
Immunohistochemistry
Thaw the sections at room temperature for at least 1 hour
Wash in PBS for 30 min twice at room temperature
Incubate in blocking buffer (5% donkey serum in PBS with 0.1% Triton X-100) for 1 hour at room temperature
Incubate the sections with primary antibodies diluted in blocking buffer overnight at 4C
Wash in PBS for 30 min three times at room temperature
Incubate the sections with secondary antibodies and DAPI for 1 hour at room temperature
Wash in PBS for 30 min three times at room temperature
Mount your slides using relevant mounting media and cover slip
Imaging
Image the sections using a confocal microscope
Analysis
Analyze the picture using ImageJ/FIJI
