Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

This study was carried out in accordance with the policies set forth in the Guide for the Care and Use of Laboratory Animals (NIH) approved by Ethic Committee of Iran University of Medical Sciences (Tehran, Iran).

1. Animals, Surgery and Specimen Preparations

NOTE: Prepare brain tissue for 3D confocal microscopic analysis.

1. Divide animals randomly into two groups: 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 surgery.
2. Anesthetize the animals with ketamine (100 mg/kg) and xylazine (10 mg/kg). Confirm proper anesthesia by lack of withdrawal reflex after pinching the toe. Use ointment on 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 midline, and −2.8 mm below dura, according to rat brain atlas⁸. For stereotaxic method please see previous publication⁹.
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 sign 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 PBS (pH = 7.4), then remove and fix brain as described in¹⁰.
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 atlas⁸ 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 with be necessary to produce Z-Stack images of complete astrocytes. Do not use the first couple 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: Over expansion of the section can disturb the morphology of the structure. Use tiny paintbrush to carefully transfer sections. Collect two to three sections on each slide.
11. Store slides in a rack in an upright position and let them dry at 37 ˚C for several hr or 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 min 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 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. Upright confocal laser scanning microscope is used to create 3D images from 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 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 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 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, the entire astrocyte including branches, cell body, and nucleus.
16. Acquire a 2D image using a 10X or 20X objective as described in Steps 3.6-3.11. This image can be used for the measurement the intensity of fluorescence immunoreactivity of antibodies, and astrocyte density (number/1,000 µm²).

4. Astrocyte Density and GFAP⁺ Fluorescence Intensity

1. Open the 20X 2D image. Click on Histo (Hisogram) on the left side of image window.
2. To calculate astrocyte density, select three consecutive visual fields under 20X objective. Count the number of astrocytes that exhibit a clear soma with minimum, overlapping branches in the fields.
3. To quantify fluorescence intensity, select a proper tool (rectangle or circle) on the footer of image window, and choose an area for the measurement. Note the mean value and standard deviation appear automatically under the image. In the current study, a 0.7 mm² rectangular area between striatum and medial blade of dentate gyrus was used.

5. Astrocyte Branches

1. To quantify the length and total number of branches, use 3D images (acquired with 20X) in the image analyzing software. Draw a line manually along the length of each branch of the selected astrocyte. Then select the measurement-tool that is specialized for measuring line-length. In order to quantify the number of branches, select the count-tool to count the number of marked items.

6. Volume and Surface Area

NOTE: Marking a structure manually in 3D image analysis software requires focus and training. Practice several times before starting an experiment.

1. To quantify the volume and surface area of the astrocyte nucleus, soma, cell body and territory, transfer the 3D images (acquired in Steps 3.1-3.16) to a 3D image analysis software such as Volocity 6.1.
2. Open the software and create a new library. Drag all images to the left gray column in the new library. Then, select one 3D image. On the right, there are different channels with different colors. Turn on one channel of interest; for example the blue channel for DAPI when measuring the nucleus. Change the brightness manually to get an optimal contrast.
3. Select the Tools menu, go to Make Volume and produce a single 3D image out of Z-stack images. Click on Properties in the Edit menu and be sure that X, Y and Z properties are presented; otherwise it is impossible to carry out 3D analysis.
4. Click on Freehand RIO tool icon in the toolbar. To measure the volume and surface area of astrocyte nucleus draw a line around the DAPI‒labeled nucleus.
5. To measure the volume and surface area of astrocyte cell body draw a line around the soma excluding branches.
6. To measure the volume and surface area of entire astrocyte (cell body including branches) draw a line around the soma following the surface of all branches.
7. To assess the volume and surface area of astrocyte territory draw a line between tips of the branches.
   NOTE: Press Delete on the keyboard to erase unwanted drawings.
8. Click on the Measurements menu at the top of the image window to create a measurement protocol; a window containing different tools opens.
9. Drag Find objects to top of the window (pane). Give a descriptive name to the Population 1, select the associated color, and click on Measure. Another window opens.
10. Choose a parameter i.e., volume or surface area. Click on OK. The data will appear in a table.
11. To save the data click on the Measurement menu and select Make Measurement Item.
12. Select A new measurement item called from the opened window. Then give a name to the data and click on OK.
13. Finally, export the data to statistics software such as Graph pad or excel program. Use t-test to analyze the data, then plot 2D graphs.