Two to four rats will be perfused with 4% paraformaldehyde and tissues of interest will be removed and stored in 4% paraformaldehyde.

Tissue Sectioning

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

Incubate overnight 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.

Set up freezing/sliding microtome freezing platform with enough crushed dry ice to fill both reservoirs. Allow platform to freeze for 15 min.

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.

Carefully shave off 30µm slices of cryoprotectant until you reach the tissue. Continue to slice through the tissue until a full section is obtained.

Cut a 3.0µm section of tissue and place it in one well of a 48 well tissue culture plate filled with 750µl 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.

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 Procedure

The tissue used for this protocol is 21 day old male Wistar rat cerebellum cross sections.

This is a two day protocol: Day One you will need four consecutive hours, followed by an overnight incubation. Day Two you will need six consecutive hours. All operations are performed at room temperature unless otherwise noted. Place the cover to the 48 well plate on during all the incubations.

1. Set up four 48 well plates by dividing each into two separate halves of six rows and four columns. Label columns 1,2,5 and 6 as “Anti-TrkB In pAb” and columns 3,4 7 and 8 as control. As you proceed through the experiment, you will be moving the tissue from the top (A1-A4) down to the bottom (F1-F4) and then moving over to the other half of the plate, starting again at the top (A5-A8) and working your way down (F5-F8) for each of the following steps. See the tables below: The table is included to avoid any confusion concerning the movement of the sections.Note that there are duplicates for the antibody and the control. The control does not receive the Anti-TrkB In pAb and therefore is the test for nonspecific reactivity (background) due to the conjugate antibody.
2. Add 750µl 1X PBS to wells A1-C4. Place three cerebellum sections each in 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 cross over between wells to a minimum.
   For each subsequent step, add the appropriate solution to the next well in the series. Do not let the sections dry out.

PLATE 1

PLATE 2

            After the final PBS wash, the tissues are mounted (see Step 17).

3. Wash the sections three times (plate 1, A1-C4) for five minutes each with 750µl 1X PBS shaking at 200 rpm on the shaking platform at room temp. 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. Incubate sections in 500µl permeabilization solution (plate 1, D1-4) for 30 minutes, shaking at 200 rpm on the shaking platform at room temp. The Triton^® X-100 permeabilizes the tissue to allow antibody penetration. The H₂O₂ quenches endogenous hydrogen peroxidase activity.
5. Wash as in step 3 (plate 1, E1-F4, A5-8).
6. Incubate sections in 500ml/well blocking solution (plate 1, B5-8) for 30 minutes, shaking at 200rpm on the shaking platform at room temp.
7. Prepare 5.0µg/ml solution of the Anti-TrkB In pAb in Blocking Solution:
8. Incubate sections in 500µl/well Anti-TrkB In pAb (5.0µg/ml) overnight in a coldroom, shaking at 200 rpm on the shaking platform (move the shaking platform into a 4°C coldroom).As a control to test for background due to the secondary antibody, omit the primary antibody and substitute the blocking solution for the overnight incubation.
9. Wash as in Step 3 (plate 1 D5-F8).
10. Prepare 2.0µg/ml donkey anti-chicken biotin conjugate: Add 10µl of conjugate to 4.990ml 1X PBS. Incubate sections in 500µl/well conjugate (plate 2 A1-4) for 30 minutes at room temp.
11. Make up Vector AB reagent now. This is a mixture of streptavidin and horseradish peroxidase that must react for 45 minutes prior to use.
12. Wash as in 8.3.2 (plate 2 B1-D4).
13. Incubate sections in 500µl/well AB reagent (plate 2 E1-4) for 60 minutes, shaking at 200 rpm on the shaking platform at room temp.
14. Wash as in Step 3 (plate 2 F1-4, A5-B8).
15. Incubate sections in 500µl DAB solution (plate 2 C5-8) for 8 minutes, shaking at 200 rpm on the shaking platform at room temp.
            DAB solution must be made up immediately before use.
16. Wash as in Step 3 (plate 2 D5-F8).
17. Apply a drop from a transfer pipet (approximately 50µl) of 100% glycerol to microscope slide, position section on the drop and coverslip. The coverslip may be permanently sealed by brushing nail polish around the edges. Permanently sealing the coverslip is done only for ease of storage and handling: it does not affect stability.
            At this point, the sections are indefinitely stable at room temperature in a covered container.

Composition of Solutions

1X PBS (200 ml): Prepare using 20 ml 10X PBS and 180 ml Nanopure Water. Store at 4°C for up to three months.

1.0% Triton^® X-100 stock in 1X PBS: To 99 ml 1X PBS, add 1.0 ml Triton^® X-100. Mix by gentle inversion. Store at 4°C for up to 3 months.

Permeabilization Solution (0.06% Triton^® X-100 + 0.1% H₂O₂) : To 9.367ml 1X PBS, add 600µl 1.0% Triton^® X-100 stock and 33µl 30% H₂O₂. Make up prior to use and throw out what is not used.

Blocking Solution/Antibody Diluent for Immunohistochemistry: 3.0% goat serum in PBS: To 48.5ml 1X PBS, add 1.5ml goat serum. Bring volume to 50ml with PBS. Unused goat serum can be stored at -20°C for future use.

Vector Labs AB reagent: Make 45 minutes prior to Step 11: Add 5.0ml of “A” to 5.0 ml 1X PBS and vortex. Add 5.0ml of “B”, vortex and let sit at room temperature for at 45 min.

DAB solution: This solution should not be made until it is ready to be used at Step 13: Add one DAB tablet to 15 ml 1X PBS. WORK IN THE FUME HOOD – Vortex until the tablet is completely dissolved. Add 20µl H₂O₂ and immediately filter through a 0.22µm filter before applying to the tissue sections.