sodium chloride
3.
1-ml Syringes and 26![$$[{3 \mathord{\left/{\vphantom {3 8}} \right.\kern-\nulldelimiterspace} 8}]$$](/wp-content/uploads/2017/04/A7657_2011_30_Chapter_IEq2.gif)
needles
needles4.
Ophthalmic ointment
5.
Commercial depilating cream
6.
Gauzes
7.
Betadine surgical scrub
8.
70% Ethanol
9.
Stereotactic frame with nonpuncture ear (Harvard Apparatus Inc.)
10.
Scalpel
11.
Ideal Microdrill (Cell Point Scientific No. 67-1000 1249)
12.
Microdrill Steel Burrs 0.5 mm (Fine Science Tools Cat. No. 19007-05)
13.
1-μl Hamilton syringe
14.
Stereotaxic injector QSI Quint essential (Stoelting Co. No. 53311)
15.
Adenovirus: Ad5CMVCre (Gene Transfer Vector Core, University of Iowa)
16.
Sterile bone wax
17.
4-0 Gut suture
18.
Surgical heat pad
2.2 BLI System Imaging
1.
Isoflurane
2.
Anesthesia chamber
3.
Xenogen IVIS 100 imaging system
4.
15 mg/ml Firefly d-luciferin
5.
Phosphate-buffered saline (PBS)
6.
1-ml Syringe
7.
26![$$[{3 \mathord{\left/{\vphantom {3 8}} \right.\kern-\nulldelimiterspace} 8}]$$](/wp-content/uploads/2017/04/A7657_2011_30_Chapter_IEq3.gif)
Needles
Needles8.
Commercial depilating cream
9.
Gauzes
10.
Living Image software
2.3 Analysis of GBM
2.3.1 Samples Collection
1.
PBS
2.
Liquid nitrogen
3.
Histology cassette
4.
4% Paraformaldehyde
2.3.2 Immunohistological Staining for GBM Markers EGFR, GFAP, NeuN, and S100
1.
Paraffin-fixed tissue
2.
Microtome (Micro HM310).
3.
Xylenes
4.
Alcohol 100, 95, 90, 80, and 70%
5.
Microwave
6.
Antigen target retrieval solution (Dao, S1699)
7.
PBS
8.
0.3% H2O2 in methanol
9.
Blocking solution (5% goat serum (SIGMA), 0.3% Triton X-100 in PBS)
10.
Mouse-on-mouse blocking reagent (Vector Labs Inc., MKB-2213)
11.
Primary antibodies: EGFR antibody (mouse mAb anti-EGFR, 31G7, Zymed Lab Inc.), anti-GFAP (Dao, Z0334), anti-S100 (Dao, Z0311), and anti-NeuN (Chemicon, MAB377)
12.
Biotinylated secondary antibody (anti-rabbit or anti-mouse from Vector Labs Inc.)
13.
Vectastain ABC kit from Vector Labs Inc.
14.
Hematoxylin (SIGMA)
15.
Coverslip
16.
Microscope
17.
Camera
3 Methods
3.1 Stereotactic Intracranial Injection of Viruses
1.
Anesthetize >3-month-old adult mice EGFRvIII/vIII; InkΔ2/3−/−; PTENlox/lox with intraperitoneal injections of 120 mg/kg ketamine and 10 mg/kg xylazine in 0.9% sodium chloride (see Notes 1 and 2).
2.
Apply ophthalmic ointment on eyes and shave the scalp using commercial depilating cream.
3.
Disinfect the shaved scalp with betadine surgical scrub and then ethanol 70% three times.
4.
Place mouse in stereotactic frame.
5.
Do a single incision from the anterior pole of the skull to the posterior ridge, skin can be retained using small clamps.
6.
Drill a 0.5-mm hole in the skull using a microdrill at the coordinate of choice. For striatum injection, we typically inject 2.1-mm rostral to the bregma, 1.0 lateral to the midline and at 2.5-mm depth to the pia surface (see Notes 3 and 4).
7.
Position the 1-μl Hamilton syringe in the hole and inject virus. Hamilton syringe is hooked onto Quint essential Stereotaxic injector and 1 μl of virus is injected at a rate of 0.1 μl/min (see Note 5).
9.
Place the mouse on a surgical heated pad and monitor animal multiple times until completely ambulatory (see Note 7).
3.2 BLI System Imaging to Monitor Tumor Growth
1.
Assess luciferase activity biweekly, ~2 weeks after virus injection.
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