Three-dimensional Imaging of Immunolabeled Astrocytes Using Confocal Microscopy

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Animals, Surgery, and Specimen Preparations

NOTE: Prepare brain tissue for three-dimensional (3D) confocal microscopic analysis.

1. Divide animals randomly into two groups: amyloid beta (Aβ)_1-40‒injection (n = 8), and Aβ_1-40‒injection with genistein treatment (n = 8); administer genistein (10 mg/kg diluted in a vehicle such as polyethoxylated castor oil) by gavage 1 hr before the surgery.
2. Anesthetize the animals with ketamine (100 mg/kg) and xylazine (10 mg/kg). Confirm proper anesthesia by the lack of withdrawal reflex after pinching the toe. Use ointment on the eyes during the surgery to prevent dryness.
3. Place animals in stereotaxic apparatus and shave head. Apply iodine solution to clean the scalp before incision and keep the operation site sterile during the surgery to reduce the risk of infection. Inject Aβ_1-40 stereotaxically (4 µl) in the hippocampus at −3.5 mm posterior to bregma, ± 2 mm lateral to the midline, and −2.8 mm below the dura, according to rat brain atlas.
4. Provide post-operative observation/care until the animals are fully conscious to ensure the comfort of the animals. Keep the animals warm during recovery, and do not return them to a cage with other animals until full recovery. Pay attention to any signs of infection in the animals after surgery.
5. Anesthetize the animals deeply by ketamine (150 mg/kg) three weeks after surgery. Perform transcardial fixation by perfusing the animals with 0.9% saline followed by 4% paraformaldehyde in 0.1 M Phosphate buffer saline or PBS (pH = 7.4), then remove and fix brain.
6. Post-fix the brains in 4% paraformaldehyde for 2-3 days at 4 ˚C.
7. Embed the tissue in paraffin blocks by use of tissue embedding equipment, and avoid under-filling or over-filling the cassette since it may interfere with correct alignment or sectioning. Pay attention to the orientation of the tissue in the paraffin block.
   NOTE: The rat hippocampus, for example, is close to posterior part of the cerebral hemisphere. Thus, the tissue should be embedded in a way that sectioning starts at posterior part of the brain. Use the Paxinos atlas as a guide to reach the desired anatomical structure.
8. Place the microtome away from air drafts or doorways since air movements make the handling of sections difficult.
9. Use sections with a thickness ≥20 µm, as this depth will be necessary to produce Z-Stack images of complete astrocytes. Do not use the first couple of sections, as they may have undesired thickness due to thermal expansion.
10. Place the sections on the surface of warm water (5 ± 2 ˚C, below the melting temperature for paraffin) just long enough to flatten the sections.
    NOTE: Overexpansion of the section can disturb the structure's morphology. Use a tiny paintbrush to carefully transfer sections. Collect two to three sections on each slide.
11. Store slides in an upright rack and let them dry at 37 ˚C for several hours or overnight (O/N).

2. Immunohistochemistry

1. Deparaffinize sections in xylene, 2 x 10 min, and rehydrate through alcohol gradient (99.8%, 95%, and 70%, 10 min each) and PBS.
2. Incubate with antibodies against glial fibrillary acidic protein (GFAP) diluted 1:1,500 in PBS containing 0.25% bovine serum albumin (BSA), 0.25% Triton X-100, and 3.5% normal serum (O/N at 4 ˚C).
3. Wash sections in PBS for 3 x 5 min.
4. Incubate with secondary alkaline phosphate-conjugated antibodies for 1h (1:100, 21˚C, diluted in PBS).
5. Wash the sections in PBS for 3 x 5 min.
   NOTE: It is important to wash the sections properly after secondary antibodies to minimize background staining.
6. Incubate sections in darkness with Liquid Permanent Red Chromogen (15-20 min). 
   Note: Prepare the solution no more than 30 minutes before use.
7. Wash sections in PBS for 3 x 5 min.
8. Finally, stain the nuclei with 4-6-diamidino-2-phenylindole (DAPI; 1:500, diluted in PBS) prior to cover-slipping. Store the slides in the refrigerator for 24-48 hr before microscopy.

3. Confocal Microscopy

NOTE: For quantitative evaluation (see below), select an astrocyte with a clearly visible DAPI-stained nucleus with a minimum of overlapping branches to reduce the risk of errors. Producing Z-Stack images is time-consuming. Be patient and do not stop during processing. An upright confocal laser scanning microscope is used to create 3D images of astrocytes.

1. Put the slide on the microscope stage and select the 63X objective.
2. Open the imaging software and click on Start System. Click on the Locate tab. Go to Assign and select GFP to adjust the proper light.
3. Open the Shutter to reflect light. Find the Reflector Revolver on the right side of Shutter and choose the color that should be reflected by astrocytes (green, red, or violet); red color (FSet20 wf) was used here.
   NOTE: Do not forget to focus the picture directly in the ocular of the microscope.
4. To find the best focus, click on All Closed, put the microscope pin in screen mode, and click on the Acquisition tab.
5. Click on Smart Setup under the Acquisition tab; a window opens. Select the proper fluorophore (i.e. DAPI and Alexa Fluor 555, in the current project) from the list. The color is automatically selected.
6. Click on Best Signal and Apply, then click on Set Exposure; the computer will then automatically set the exposure parameters.
7. For optimizing the image, go to the Channels window. Unmark Track2, highlight Track1 and click on Live. Focus on the image if needed.
8. In the Channels window adjust the following keys; click on 1AU to automatically optimize the pinhole. It is very important to do this step otherwise the confocal images will be non-optimal. Adjust Gain to have the best intensity (keep this setting below 800 to reduce extra noise). Finally, set the Digital Gain between 2 and 3.
9. Unmark Track1, click on Track2, and repeat Step 3.8 for Track2. Then stop Live.
10. Go to the Acquisition Mode window. Improve the image by optimizing the Frame Size and Averaging. In order to change frame size go to X*Y and select 1,024 x 1,024. Choose a slow Speed to create a better image.
11. Go to Averaging and select a Number ≥4. This averaging value indicates the number of scans that will be averaged to produce the acquired image. Now, click on the Snap button and save the captured 2D image.
12. For 3D imaging, mark the Z-Stack item under Smart Setup, causing the Z-Stack window to open.
13. Set the first and last positions for the Z-Stack, i.e., the depth of the cell. First, click on Live. Then use the focus drive of the microscope to focus on the uppermost position of the astrocyte in the tissue, where the Z-Stack is to start. Click on Set First. Then, focus down to the lowest position of the astrocyte in the tissue, where Z-Stack scanning should be stopped. Click on Set Last. Then stop Live.
14. Choose the Interval; 1.01 µm is used in the current study; choosing interval can be done by clicking on Optimal to set the number of slices.
15. Click on Start Experiment. Save the images once the scan is complete. This image will be used for the measurement of the following parameters: surface area and volume of astrocyte territory, as well as the entire astrocyte, including branches, cell body, and nucleus.