Remove rat spinal cords and place in ice-cold 10% formalin in 50ml tube Store the spinal cords in their original tube at 2-10°C overnight. Continue with Tissue Sectioning Step 1 for sectioning of the tissue. This is a very clean IHC and no other blocking is needed than that provided by the Triton^® X-100 in the antibody diluent.

Tissue Sectioning

You will need approximately 2-3 hours to section enough material for one experiment. This will require 2 days:

1. Incubate overnight (12-14hr) in cryoprotectant at 4°C in a 50 ml tube or similar vessel. Tissue should not be stored in cryoprotectant for longer than the overnight period. Cryoprotectant: add 30g sucrose to 50 ml PBS. Bring volume to 100ml and filter sterilize using a 150ml filter unit (0.2µm filter). Store at 4°C up to 1 month. Filter sterilize before each use.
2. Set up freezing/sliding microtome freezing platform with enough crushed dry ice to fill both reservoirs. Allow platform to freeze for 15 minutes.
3. Pool cryoprotectant in the center of the platform to form a base for the tissue. Place tissue in proper orientation on top of the base and pool cryoprotectant around and on top of tissue. Allow to freeze for 20 min.
4. Carefully shave off 30µm slices of cryoprotectant until you reach the tissue. Continue to slice through the tissue until a full section is obtained.
5. Cut a 3.0µm section of tissue and place it in one well of a 48 well tissue culture plate filled with 750 ml 1X PBS. The sections are easy to pick off the blade with a paintbrush and will unfold neatly in the 1X PBS. Place five sections per well. For each experiment you will need approximately 20 sections.
6. The sections should be stored in the tightly wrapped 48 well plate at 4°C until used. It is best to use the sections within three days. After this time it is possible to lose some signal.

Immunohistochemistry (IHC)

This is a two day protocol: DAY 1 you will need approximately one hour, followed by an overnight incubation. DAY 2 you will need approximately three consecutive hours.

DAY 1

Note: cover plate with supplied cover during all incubation steps.

1. Set up one 48 well plate as shown in the template below.
   As you proceed through the experiment, you will be moving the tissue from the top down to the bottom until you get to row F. In the template below, it tells you where to go after you have reached the bottom of the plate.
   
   

2. Add 750µl PBS to wells A1-C4. Place four sets of spinal cord sections into wells A1-A4. A disposable culture loop works well for this. The loop can be reused throughout the entire experiment if it is dipped into a beaker of Nanopure water before being used in the next well. This dipping to wash loop is critical to keep contamination between wells to a minimum.
3. Wash the sections three times for five minutes each with 750µl PBS (A1-C4) shaking at no more than 200rpm on the shaking platform. The sections are “washed” by moving them from one well into a new well. This is a minimum wash: the sections can wash for longer times with no deleterious effects.
4. Prepare 1.0µg/ml solution each of the Anti-VAChT pAb in VAChT Antibody Diluent
5. Place 500µl diluted Anti-VAChT pAb in D1, D2, and 500µl VAChT Antibody Diluent alone in D3 and D4.

Move the tissue sections from C1-4 into their respective well and incubate overnight in a coldroom, shaking at no more than 200 rpm on the shaking platform (move the shaking platform into a 2-10°C cold room).

END OF DAY 1

DAY 2

2. Add 750µl of PBS to wells E1-4, F1-4, and A5-8. Transfer sections from D1-4 into their respective wells in row E, etc. Wash as in Step 3 above.
   
3. Prepare 6.0µg/ml donkey anti-goat Cy3™ conjugate: Add 12µl of conjugate to 2.988 ml 1X PBS.
   
   Note: Although Cy3™ is very robust fluorochrome, it can begin to loose some of its signal if overexposed to light. From this point onward, try to limit the exposure of the Cy3™ (and therefore the sections) to light. This can be done by placing a small box over the 48 well plate while it is incubating. You will have to mount the tissue later under the dissecting microscope lights. This is unavoidable but use the lowest possible setting for light intensity to reduce the amount of light.
4. Place 500µl of donkey anti-goat Cy3™ conjugate in wells B5-8. Transfer sections from A5-8 into their respective wells. Incubate tissue sections for 30 minutes in a 37°C incubator.
   Note: It may not be possible to place the shaking platform in the 37°C incubator. If it is not, gently swirl the plate every 5 minutes to keep the sections from sticking together.
5. Add 750µl of PBS to wells C5-8, D5-8, and E5-8. Transfer tissue sections from B5-8 into their respective wells in row C, etc. Wash as in step 3 above.
   
6. Label a microscope slide with the appropriate information for each sample. Place a drop (approximately 50µl) of Vectashield + DAPI in the center of each microscope slide.
   
7. To the appropriate slide, transfer all 3 tissue sections from each sample to the drop of Vectashield + DAPI using a disposable culture loop. Flatten the sections out using 18 ga needles while viewing under a dissecting scope.
   
8. Place a coverslip over the sections, invert the slide on a paper towel and gently push down to remove air bubbles and to further flatten the section.
   
9. Seal the coverslip now or after analysis and photography by “painting” around it with fingernail polish. Allow to dry approximately one hour. This step is included since this is the best way to store the slides indefinitely. It does not affect the results and can be done after analysis and photography to save time: most people would like to look at their results right away.

Analysis and Photography of the Stained Tissue Sections

View the specimen using the 20X objective using the Cy3/5 filter to get the best view of the area of interest. VAChT positive areas will be localized to the ventral motor neurons in the gray matter of the rat spinal cord. Negative control should show no specific label.

Compostion of Solutions

Cryoprotectant: Add 30g sucrose to 50 ml PBS. Bring volume to 100ml and filter sterilize using a 150ml filter unit (0.2µm filter). Store at 4°C up to 1 month. Filter sterilize before each use.

1X PBS (200 ml): Prepare using 20ml 10X PBS and 180ml Nanopure Water. Refrigerate for up to 6 months.

1.0% Triton^® X-100 Stock: To 99 ml PBS, add 1.0ml Triton^® X-100. Mix by gentle inversion. Refrigerate for up to 6 months

VAChT Antibody Diluent (50ml of 0.3% Triton^® X-100 in PBS): To 35ml 1X PBS, add 15ml 1.0% Triton X-100 stock. Make fresh prior to use.