Materials

 Differentiation of mouse embryonic stem cells and chick muscle cells

DFK10 medium, 60mm Petri dish, sonic hedge hog (Shh), retinoic acid (RA), embryonic mouse stem cells, chick myoblast cells, 12mm poly-D-lysine/lamanin coverslip, 35mm Petri dish, incubator, centrifuge, 24-well tissue culture plate, 12-well tissue culture plate, glass disks, pipettes, pipette tips

 Immunohistochemistry

3.7% formaldyhyde in PBS, cryprotectant (10% sucrose in 50mM sodium phosphate buffer), normal goat serum (NGS), normal donkey serum (NDS), disposable tubes, Petri dishes, monoclonal antibodies (Alpha 1C, 5E NCAM), polyclonal antibodies (Cy3, Alex fluror 350), phosphate buffer (PB), phosphate buffer with Triton-X (PBS-T), phosphate buffered saline (PBS), glass slides, mounting media, 0 refraction cover slips, Zeis Axiovert 200 with an AxioCam Digital microscope, Axiovision computer program,  sealant, silicone-filled syringe

 Methods

 C2C12 Myoblast Differentiation

A cell line of chick myoblast cells were differentiated into muscle tissue.  The C2C12 cells were thawed following storage in liquid nitrogen.  The cells were rinsed through a process of centrifugation, supernatant replacement and re-suspension of the cell pellet with fresh medium.  This rinsing process was repeated. 

 The C2C12 cells were plated in a 24 well plate containing 12mm round coverslips coated with poly-D-lysine/lamanin.  The C2C12 cells were plated in 500μl of C2C12 cell medium supplemented with 10% normal horse serum.  The cells were fed after the first 3 days of plating and then once every two days thereafter.  The cells were allowed 10 days before the population was sufficiently differentiated into muscle.  At this stage the muscle cells were ready to be plated upon.

 ES Cell Differentiation

Mouse stem cells were harvested from embryonic fluid.  The embryonic stem (ES) cells were suspended in 5ml of DFK10 medium within a 60mm Petri dish for two days prior to treatment with sonic hedge hog (Shh) and retinoic acid (RA).  The ES cells (which have now formed embryoid bodies (EBs)) were induced to differentiate into motor neurons (MNs) through treatment with 5μl of Sonic hedgehog (Shh) and 5μl of Retinoic Acid (RA) for a final concentration of 1μM for both Shh and RA.  Following this treatment of Shh and RA, the EBs were left for 5 days in order differentiate.  At day 5 the EBs were plated onto cultured chick muscle cells.  For plating the coverslip with cultured chick muscle cells was transferred to a 35mm Petri dish in 3mm of DFK10 medium.  Then, 2 to 3 EBs were pipetted atop of the muscle tissue and left to adhere to the tissue for an hour.

 Immunohistochemistry

The coverslip with mouse EBs and chick muscle cells was placed in a 12-well tissue culture plates andwere werewwwer fixed for one hour with incubation at room temperature in 3.7% paraformaldehyde in PBS.  The tissue waswere  then placed in cryoprotectant in 10% sucrose in 50mM sodium phosphate buffer (PB.)  The tissue sample was washed three times in PBS with 3% Triton (PBS-T).  A blocking procedure was then performed for two hours at room temperature using 3% Normal Donkey Serum (NDS) in a PBS-T solution.

 The cells were incubated at 4º Celsius overnight in Alpha 1C monoclonal rabbit antibodies at a concentration of one to five thousand in a solution of 1% NDS in PBS-T to recognize the Alpha 1C subunits of any calcium channels present.  After the incubation, the tissue was washed three times in PBS and then incubated at room temperature for two hours in a solution of Cy3-conjugated polyclonal donkey anti-rabbit antibodies at a concentration of one to two hundred fifty in 1% NDS in PBS-T. The tissue was rinsed once in PB. 

 The tissue sample was placed on a glass slide and a few drops of the mounting media were placed upon it.  A wall was created around the tissue sample with silicone, and a zero refraction cover slip was placed on the silicone wall.  The sides of the cover slip were sealed.  The slides were studied and photographed using a fluorescent microscope.  This procedure was repeated on three more occasions.   

A negative control was set up by plating a coverslip confluent with chick muscle cells in a 12-well tissue culture plate.  The muscle cells were fixed with incubation at room temperature in 3.7% paraformaldehyde PBS for one hour.  They werewere  then placed in cryoprotectant of 10% sucrose in 50mM sodium phosphate buffer (PB.)  The tissue sample was washed three times in PBS with 3% Triton.  A blocking procedure was then performed for two hours at room temperature using 3% Normal Donkey Serum (NDS) in a PBS-T solution.

 The cells were incubated at 4º Celsius overnight in Alpha 1C monoclonal rabbit antibodies at a concentration of one to five thousand in a solution of 1% NDS in PBS-T to recognize the Alpha 1C subunits of any calcium channels present and in 5E NCAM monoclonal rabbit antibodies at a concentration of one to fifty to label the muscle fibres.  After the incubation, the tissue was washed three times in PBS and then incubated at room temperature for two hours in a solution of Cy3-conjugated polyclonal donkey anti-rabbit antibodies at a concentration of one to two hundred fifty and of Alex fluror 350-conjugated polyclonal donkey anti-rabbit antibodies at a concentration of one to two fifty in 1% NDS in PBS-T. The tissue was rinsed once in PB. 

 The tissue sample was placed on a glass slide and a few drops of the mounting media were placed upon it.  A wall was created around the tissue sample with silicone, and a zero refraction cover slip was placed on the silicone wall.  The sides of the cover slip were sealed with sealant.  The slides were studied and photographed using a fluorescent microscope.  This procedure was repeated on three more occasions.