2.1. Animals

2.2. Preparation of Brains for Immunohistochemistry

• Ketamine (10%, 1 ml/kg) and xylazine (2%, 0.5 mL/kg).
• Mini-peristaltic pump for intracardial perfusion of small animals.
• Phosphate-buffered saline (PBS, 10X): 137 mM NaCl, 27 mM KCl, 14 mM Na2HPO4, and 43 mM KH2PO4. Bring total volume to 1 L with deionized water (see Note 1). Adjust pH to 7.4. Sterilize by autoclaving.
• 4% Paraformaldehyde (PFA) in PBS 1X, pH 7.4.
• Cryoprotective solution I (for brains after perfusion): 30% sucrose in PBS 1X.
• N-methylbutane.
• Laboratory dissection tools for brain preparation and extraction.

2.3. Preparation of Slices for IF-IHC

• Cryostat.
• 12-well plates (see Note 2).
• Optimal cutting temperature (OCT) compound to fix brains for cutting in the cryostat.
• Cryoprotective solution II (for slices after cutting): 25% ethylene glycol, 25% glycerol in PBS 1X pre-cooled to -20 °C.

2.4. Immunofluorescent Staining

• 24-well plates.
• Washing buffer: PBS 1X.
• Blocking/permeabilization buffer: PBS 1X, 0.5% Triton X-100 and 5% normal goat serum (NGS).
• Primary antibody buffer solution: PBS 1X, 0.5% Triton X-100 and 5% normal goat serum (see Note 3).

1.

Secondary antibody solution: PBS 1X.

2.

Primary antibody

• rabbit monoclonal anti-GFAP antibody (1:200, Cell Signaling D1F4Q XP) to label the cytoskeletal structure of astrocytes.

3.

Secondary antibody

b.

goat-anti rabbit Alexa Fluor 488 (1:1000, Invitrogen).

4.

Round-shaped glass plate to mount slices on slides.

5.

Pre-cleaned microscope slides (Superfrost, e.g. Fisher Scientific).

6.

Roti® Mount FluorCare DAPI.

7.

Microscope cover glass (22 × 60 mm, e.g. Fisher Scientific, Pittsburgh, PA).

8.

Pipettes, brushes, and other general laboratory equipment.

9.

Horizontal shaker.

10.

Confocal microscope (CLSM, Zeiss LSM 980).

11.

ImageJ Software (http://www.imagescience.org).

3.1. Preparation of Brains for Immunofluorescent-Immunohistochemistry (IF-IHC)

• Following behavioral experiment (2), anaesthetize the animal by administering intraperitoneally (i.p.) a mixture of ketamine and xylazine (see Note 4).
• Transfer the animal on a perfusion tray, fix their four paws with tape or pin so that nothing moves during the procedure.
• From this step on, operate within a fume hood (see Note 5).
• With the help of forceps, hold the skin of the animals above the chest and with scissors open it by cutting a small hole below sternum. Carefully cut laterally just under the diaphragm. Then carefully cut the diaphragm along the entire length of the ribcage. Clamp the sternum with a pair of hemostats and lift it up to fully expose the chest cavity with heart and lung.
• Carefully tear off pericardial sac to enable to have a clear view of the heart.
• Take the apex of the heart with your thumb and fore finger or a good pair of tissue forceps. Insert the perfusion needle into left ventricle and hold it there with a pair of hemostats and make a small incision on the right atrium using fine scissors.
• Let the cold (4 °C) PBS 1X flow at a steady drip of 19ml/min (see Note 6) until the liver turns to a pale colour, which indicates a good flush of arteries/veins from blood with PBS 1X.
• Change the PBS 1X to cold PFA 4% and continue perfusing the animal for 3-4 minutes (see Note 7) until all paws and liver become whitish and blenched, respectively.
• Remove needle and clamps (hemostats) and decapitate the head with a pair of scissors.
• After decapitation, open the skull and remove the brain.
• Place each brain in a 50-ml falcon tube for post-fixation overnight (o.n.) in 4% PFA at 4 °C.
• After one day, transfer the brains to Cryoprotective solution I. Let the brains in this solution at 4 °C until they sink.
• After sinking of brains, wipe them from excess of liquid and flash-frozen N-methylbutane pre-cooled in dry ice before placing them in a piece of aluminium foil (or other foil) labelled with the number of the respective animal.
• Place brains at -20 °C or -80 °C until ready for cutting and staining (see Note 8).

3.2. Preparation of Free-Floating Sections for IF-IHC

• Take out brains from the freezer and allow them to equilibrate to the cutting temperature of the cryostat for about 20-30 minutes (see Note 9).
• Prepare a 12-well plate with about 2 ml of cryoprotective solution II at -20 °C per well.
• Prepare the brain holder of the cryostat with a thin layer of OCT compound and immerge the brain into it in a vertical position (olfactory bulbs towards you) (see Note 10), surround it at the base with additional OCT and keep it in this position until the OCT is completely frozen.
• Place the block in the correct position in the cryostat and start cutting at 30-40 µm (see Note 11)
• Collect the slices using a thin brush that has been previously dampened with a small drop of cryoprotective solution II to ease the peeling of the slice (see Note 12). Place the slice into the 12-well plate following the series for each brain region.
• Once the brain has been cut to the desired extent, remove it from the block and put it back into the foil for further cutting, if necessary.
• Put the plate at -20 °C until proceeding with the staining (see Note 13).
• Procedure for the double IF-IHC.
• Remove the plates from the freezer and, using a thin brush, select a series of LS region for staining from each brain.
• Transfer slices in a 24-well plate with about 1 ml of PBS 1X per well and let them wash on a horizontal shaker for 20 minutes at room temperature (RT). Discard the buffer and repeat this step other two times to allow a wash-through of the cryoprotective solution II (see Note 14).
• Remove the buffer and incubate slices in the blocking/permeabilization solution for 1 hour (hr) at RT.
• Remove the blocking solution and incubate slices in the antibody solution containing the primary antibody o.n. on a horizontal shaker at 4 °C (see Note 15).
• Remove the antibody solution and wash slices 3 times with PBS 1X for 20 minutes each, on a horizontal shaker at RT.
• Remove the antibody primary solution (see Note 16) and incubate slices the with secondary antibody solution for 2 hrs on a horizontal shaker at RT (in a closed box or covered with aluminium foil to keep the fluorescent dyes protected from light).
• To keep the fluorescent dyes protected from light, from this step on operate in a dimly lit condition.
• Remove the secondary antibody solution and wash slices for three times with PBS 1X for 20 minutes each at RT (keeping always the plate protected from light).
• Put slices from one series in a glass petri dish filled with PBS 1X and mount them on a slide with the help of a thin brush to gently drift them.
• Let slices to air-dry inside a closed box or under an aluminium foil.
• Put few drops of mounting medium containing DAPI on slides and cover them with a microscope cover glass (see Note 17).
• Keep sections in a refrigerator until microscopic analysis.

3.3. Confocal Microscopy

• Visualize and select the region of interest for image capture with a confocal laser scanning microscopeusing a 20x objective.
• Switch to a 63x oil-immerged objective and capture three z-sections per each brain, each 30 µm-thick, with a 0.5 µm/z section interval, and acquired at 1024 × 1024 resolution.
• Save files in the existing proprietary CZI file format (.czi) file format.

3.4. Morphological Analysis

• To analyze astrocyte morphology open the file using ImageJ Software with the FIJI update.
• Emphasize details within the stack, by “flattening” the 3D stack through Z-projection at maximum intensity.
• To facilitate visualization of the astrocyte soma, generate a merge channel with GFAP and DAPI, represented in green and blue, respectively.
• Within the picture frame, select astrocytes whose structure is fully captured. Analyze the number of primary processes, the length of the longest process, and the area covered by each astrocyte (see Note 18).
• To do this, first visualize the soma of the astrocyte, and count the number of primary processes. Then, use the segmented line tool to measure the length of one of the longest processes. Finally, measure the total area covered by each astrocyte using the polygon selection tool.
• The measurements of the length and the area coverage is expressed as arbitrary units (a.u.).

3.5. Statistical Analysis

• For each brain (animal) and parameter considered, calculate the average among the three pictures. Perform a normality test using Shapiro-Wilk test to ensure the data follows a normal distribution.
• If the data is normally distributed, use an unpaired t-Test of the two groups of interest. Use the Mann-Whitney test if the data is not normally distributed.