TITLE: Rat organotypic cultures for AAV-mediated vital labeling of the extracellular matrix
AUTHORS: Federico N. Soria, Mario Fernandez-Ballester

[DESCRIPTION]
In this protocol we describe the preparation and maintenance of rat organotypic cultures (cortico-striatal) for AAV-infection and imaging of the extracellular matrix. The AAV, in this case, induces the expression of a exportable hyaluronan-binding protein (the HA-biding domain of Neurocan) fused with GFP, that allows vital labeling (and hence live imaging) of the extracellular matrix. The organotypic cultures can be maintained for several weeks, allowing for repeated imaging and longitudinal studies.

[STEPS]
SECTION: Culture preparation
1. Prepare a 6-well culture plate with Millipore 0.4 µm culture inserts placed on top of 1 mL of organotypic medium (see Materials) per well. Incubate it at 37 °C and 5% CO2 to warm up.

 

    
SECTION: Culture preparation
2. Prepare a p100 and p60 petri dishes with 25 mL and 5 mL, respectively, of   and keep them on ice .


SECTION: Culture preparation
4. Sacrifice P5-P7 rat pups by rapid decapitation and extract the brain without damaging the cortex. 
SECTION: Culture preparation
5. Put the brain on a filter paper (to remove excess liquid) and cut through the midline to separate the cerebral hemispheres. Remove the midbrain, separating it from the cortex with a scalpel.
SECTION: Culture preparation
6. Place both hemispheres in the same holder plate, with the medial face downward. Hold the plate with both hemispheres perpendicular to the blade (coronal sections) and section the brain at 350 um width.
SECTION: Culture preparation
3. Prepare the McIllwain Tissue Chopper (see Materials) with a fresh blade and clean holder plates.


SECTION: Culture preparation
7. Pour the sliced brain carefully into the p100 petri dish with HBSS on ice . You may use a Pasteur pipette to moisten the brain and slide it more easily.

SECTION: Culture preparation
8. Separate slices under the magnification microscope with a couple of spatulas (preferentially plastic-ones, which can be custom crafted cutting the back of a Pasteur pipette). Use a blade and clean cuts to remove unwanted regions (such as hippocampus or SVZ). Transfer the slices into the p60 petri dish (you can use an inverted glass pipette with a suction rubber bulb).
 


 
SECTION: Culture preparation
9. Under the laminar flow hood, transfer the selected slices (undamaged slices from the preferred rostro-caudal levels) to preheated culture inserts with medium (Step 1). Each insert can only hold two cortico-striatal slices. Remove excess dissection buffer from the top of the insert with a sterile tip.
 

From now on, the cultures should always be handled in sterility.
SECTION: Culture preparation
10. Incubate the culture at 37 °C and 5% CO2. 
 


SECTION: Maintenance and AAV-infection
11. At DIV1 (24h after culture preparation), replace the feeding medium with 1 mL of the same medium but adding antimitotic   4,4 um per well.

 

SECTION: Maintenance and AAV-infection
12. At DIV3, replace the AraC-containing medium with fresh medium. Then add 1 uL of the virus of choice (in our case, AAV-Ncan-GFP 1012 vg/mL) over the slice. 
 

 

SECTION: Maintenance and AAV-infection
13. To maintain the culture, change medium three times a week (1 mL per well).
 

SECTION: Imaging
14. Imaging can be performed in two ways:
IN-PLATE: The insert is not removed from the plate, and the imaging is performed through the plastic. This is usually done in widefield setups, with low magnification. It is not optimal in terms of resolution, but it is very useful for repeated imaging (longitudinal studies) without compromising sterility, since the plate lid is not removed during the imaging process.
OFF-PLATE: The insert is removed from the plate and placed in a smaller petri-dish or similar holding chamber. Imaging is usually done from above, in upright microscopes (objective on-top), with water-based objectives. Since CO2 is usually not available, a buffer medium with HEPES is recommended. Phenol-red should be avoided as well, since it interferes with fluorescence. This type of configuration is typically used for confocal or 2-photon imaging of the slices. Repeated imaging can be done but special attention should be paid to ensure cleanliness during the procedure (clean objective with 70% EtOH, change medium with fresh antibiotic/antimitotic immediately after imaging, keep an eye for contamination over the following days).