Twelve Hour Golgi-Cox Staining of Planarians
LOVELL, B., AGNEW, J., STILLAR, A., WEEKS*, A.C.W., & SAARI*, M.J.
NURON Labs, Nipissing University, North Bay, Ontario, Canada, P1B 8L7
Introduction
Despite being simple organisms, the delicate
tissue of small freshwater flatworms can be
difficult to stain with consistency, especially their
nervous tissue. We have developed a method of
Golgi-Cox staining to utilize on planarians
(Dugesia tigrina) based on a combination of
methodologies commonly used in mammalian
brains. Due to the delicate nature of planarian
epithelium, traditional and “rapid” Golgi-Cox
methods, which take days or weeks, tend to
damage the tissue making it nearly impossible to
get consistent, high-quality results. Our method
is
simple, consistent, and relatively quick;
optimal staining can be completed in just under
16 hours.
Methodology
Fixation
Rinse x 2
Golgi-Cox
Results
• 6% Gluteraldehyde under coverslip
• 10 minutes
Nerve cord
Eye spot
• PBS, pH 7.4
• 10 minutes each
• 5% Potassium dichromate, 5% mercuric
chloride, & 5% potassium chromate
• 12hrs at 37◦C
• Distilled H2O x 2, 5 minutes each
• 50% EtOH, 5 minutes
Dehydration • 5% Ammonium Hydroxide, 5 minutes
Figure 2: Transverse neural crossings, captured using light
microscopy at 100x magnification
Step 1
• Distilled H2O x 2, 5 minutes each
• 5% Sodium thiosulfate, 10 minutes, dark
Dehydration room
Step 2
• 70%, 80% (x2), 95% (x2) EtOH, 5 minutes
each
Dehydration • 99% 1-Butanol, 5 minutes
Step 3
http://bio1152.nicerweb.com/Locked/me
dia/ch49/nervous-flatworm.html
Figure 1: Planarian central nervous
system
Figures 3 & 4: Neural tissue, captured using light microscopy at 1000x
magnification
Conclusions
• This relatively straightforward and quick protocol permits
visualization of neural tissue in the intact planarian.
• The ability to visualize neural tissue in these worms provides a
critical tool for the examination of neurally-mediated behavioural
plasticity in this simple nervous system.