الفهرس 
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Abstract
The perfusion step was performed after 3 weeks for the AAV5-EYFP Cre dependent virus while 42 and 28 hours for Rabies virus. Animals were deeply anaesthetized intraperitoneally with a mixture of ketamine and xylazine as previously described. Then, 100 ml 4% paraformaldehyde (PFA) in 0.24 M phosphate buffer (PB), pH 7.2 -7.4 was injected intracardiac. The brains were removed and incubated in the same fixative solution (PFA 4%) for one hour under agitation at room temperature then rinsed 3 x 30 minutes in 0.12 M PB. The brains were incubated overnight at 4°C in a 20% sucrose solution in 0.12 M PB, pH 7.2-7.4. The sucrose replaces the water molecules inside the cells preventing water crystallization under -80 °C which causes a rupture of the plasma membrane.
Cryopreservation
The brains were embedded in an Optimal Cutting Temperature (O.C.T) compound which is a medium for frozen tissue specimens to ensure optimal cutting temperature. Then frozen brains were preserved in -80°C until being cut into 40 μm thickness using a cryostat (Institute des Neurosciences de la Timone INT, Aix-Marseille University, France). This cryostate was adjusted at specimen and chamber temperature; -17 and -20 respectively. Each free floating coronal section was kept in an eppendorf containing a cryoprotection solution, pH 7.2-7.4 and stored at - 20°C until histological investigations.
4.2.2 Histological investigations
Cresyl Violet Staining (Nissl Staining) The Nissl staining is a classical staining method used traditionally on nervous tissues in order to determine the general histological characteristics of tissues within the rostral-caudal directions of the brain. The principle of this technique is to stain negatively charged nucleic acids like DNA and RNA with a basic dye such as cresyl violet. In order to identify the levels of the SuML and RSC, each 10 sections were stained with cresyl violet as described below. Sections were rinsed 30 minutes in 0.12M PB (pH 7.2-7.4), followed by 2 x 30 minutes in 0.02M Potassium phosphate buffer saline (KPBS; pH 7.2-7.4) under agitation at room temperature. Then, sections were mounted on gelatin coated slides (Thermo-Fischer scientific, Waltham, MA, USA) and dried overnight at room temperature away from light.
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On the next day, slides were immersed for 2 x 10 minutes in 95% alcohol followed by 15 minutes in Chloroform-Ether solution as a de-fatting step. Then, these slides were rehydrated for 2 minutes in each of the following descending gradient of alcohol concentrations 95%, 80% and 70% before being stained for 1 minute in Cresyl violet dye (heated until 60°C). Slides were rinsed 10 times moderately in dist.H2O to remove the excess of the stain, and then immersed in Acetic Formalin up to 6 minutes for better stain differentiation. The slides were dehydrated again for 2 minutes in each of the following ascending gradient of alcohol concentrations: 80%, 95%, 95%, 100% and 100%, followed by 3 times in Histolemon solution. Finally, sections were covered by EuKit medium (Chem lab, Zedelgem, Belgium).
4.2.3 Immunohistological investigations
Endogenous Fluorescence detection In case of AAV5-EYFP Cre dependent anterograde virus, it was necessary to verify the endogenous expression of EYFP so that, the adjacent sections for the cresyl violet sections were mounted on gelatin coated slides (superfrost) and dried overnight at room temperature. On the next day, sections were coverslipped with flouromount medium (Electron Microscopy Sciences, Hatfiled, PA, USA).
Immunohistochemistry procedure The principle of the immunohistochemistry is the detection of certain antigens which are present in a tissue or a cellular structure by applying specific antibodies against these antigens. Rabies virus detection Detection of rabies virus expression level was achieved by immunohistochemistry experiments on adjacent coronal sections to the cresyl violet stained sections as detailed below. The protocol of immunohistochemistry was lasting for three days and all the steps were performed under agitation at room temperature. Mouse On Mouse kit (M.O.M, Vector Laboratory, CA, USA) was used in order to decrease non-specific staining due to the use of mouse monoclonal primary antibodies on mouse tissue. On the first day sections were washed 3 X 30 minutes in 0.12M PB, pH7.2-7.4 followed by incubation for 30 minutes with 1% Hydrogen Peroxide (H2O2; Acros organic) diluted in 0.12M PB. Sections were washed 1 x 30 minutes in 0.12M PB, followed by 2 x 30 minutes in 0.02M KPBS, pH 7.2-7.4. Then, sections were pre-treated by incubation for one hour with 2 drops of M.O.M mouse IgG blocking reagent in 0.02M KPBS + 0.3% Triton X-100 and then they were washed 2 x 5 minutes in 0.02M KPBS. Sections were incubated again for 15 minutes in
Materials and Methods
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M.O.M diluent solution (600 μl of protein concentrate solution in 7.5 ml 0.02M KPBS + 0.3% Triton X-100. Sections were incubated overnight in a humid chamber with the primary antibody anti-RV (1:5000, Raux et al., 1997) diluted in the previously prepared.