Rat spinal cords were purchased from PelFreez. The spinal cords are freshly removed at PelFreez and placed into 10% formalin. They are then shipped overnight at 4°C. The spinal cord is cut into smaller pieces (~5 mm long) with a razor blade and post fixed overnight in the same fixative. The following day (tissues may be stored indefinitely in fixative) the tissues are rinsed a couple of times in PBS and cryoprotected overnight in 30% sucrose in PBS. The following morning, 30µm sections are cut on a Zeiss freezing/sliding microtome and placed in PBS. Sections should be used within one week of cutting.

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 ~2 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 two 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-Cyt c mAb” 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-Cytochrome c mAb but you could use the Anti-b-Galactosidase mAb therefore is the test for nonspecific reactivity (background) due to the conjugate antibody.
2. Add 750µl 1X PBS to wells A1-C4. Place three spinal cord 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 (Made up on Day 1)

PLATE 2 (Made up on Day 2. Protect from light)

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 [0.3% Triton^®X-100 in PBS] (plate 1, D1-4) for 30 minutes, shaking at 200 rpm on the shaking platform at room temp.
5. Wash as in step 3 (plate 1, E1-F4, A5-8).
6. Incubate sections in 500µl/well blocking solution[3% BSA in PBS or 3-5% Donkey Serum in PBS] (plate 1, B5-8) for 30 minutes, shaking at 200rpm on the shaking platform at room temp.
7. Prepare 1.0µg/ml solution of the Anti-Cytochrome c mAb in 0.5%BSA in PBS or 1% Donkey Serum in PBS. For a control make the same dilution with the Anti-b-Galactosidase mAb (Cat. #Z3781).
8. Incubate sections in 500µl/well Anti-Cytochrome c mAb (1.0µg/ml) overnight in a coldroom, shaking at 200 rpm on the shaking platform (move the shaking platform into a 4°C coldroom)

Day 2

9. Wash as in Step 3 (plate 1 D5-F8).
   Protect plate from light from this point onward
10. Incubate in donkey anti-mouse Cy^®3 conjugate [Jackson ImmunoResearch] diluted 1:250 (5µg/ml) 60 min
11. Wash as in Step 3 (plate 2 B1-D4).
12. Apply a drop from a transfer pipet (approximately 50µl) of VectaShield + DAPI to microscope slide, position section on the drop and coverslip. The coverslip may be permanently sealed by brushing nail polish around the edges.