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which prepared the animals for the final behavioral test to C-terminal fragment-β and soluble amyloid precursor
determine the retention of memory to find the platform. protein α analysis
Two days following the nonspatial pretraining, the hidden C-terminal fragment-β (CTF-β) is generated from
platform was placed in the center of one quadrant of the APP by β-secretase in the amyloidogenic pathway,
pool, the animal was released facing the pool wall in a and soluble APP (soluble amyloid precursor
random fashion, the time was recorded (latency period), protein) is generated from APP by α-secretase in the
and the distance traveled to reach the platform was nonamyloidogenic pathway. Western blots measured
measured using video recording (Smart Video Tracking CTF-β and sAPPα brains of transgenic mice, using the
System; SD Instruments). same amount of protein per gel lane, performed as
previously described. [27,28] CTF-β was determined in the
On the day after the last training session, the platform pellet fraction from the brain extract (antibody 8717,
was removed, and a spatial probe test conducted. sigma) and sAPPα was assessed in the supernatant
Each mouse was allowed to search for the platform fraction from the brain extract (antibody 6E10,
for 60 s (memory retention) and the percent signet laboratories). Relative amounts of CTF-β and
time spent in quadrant where the platform was sAPPα were measured by densitometry and results
located (northeast (NE) quadrant) and in the outer were expressed as a percentage of the mean levels
annular area were determined. of CTF-β and sAPPα of the control groups (without
protease gene knockouts). Control β-actin western
Brain amyloid plaque blots (antiβ-actin from Cell Signaling Technology)
Amyloid plaque load was assessed in brain sections (10 was conducted to monitor equal loading of the same
from each mouse) as we have described previously, amounts of samples (20 μg protein) in each gel lane.
achieved by immunohistochemical staining for Aβ (Aβ
antibody 10D5, Elan pharmaceuticals). [27,28] Brain Immunohistochemistry staining
tissues were fixed in 4% paraformaldehyde and then Cryosections of the right brain hemispheres were
in 4% parformaldehyde and 30% sucrose for 24 h and washed 3 times (5 min/wash) with Tris-buffered
each at 4 °C. Tissues were washed in buffered saline saline (TBS) (pH 7.4) buffer, followed by washing
and transferred to an optimum cutting temperature 1 time with 0.1% Triton X-100-TBS buffer for 5 min.
medium. Cryosections were cut and blocked with Sections were then incubated in 3% H O and TBS
2
2
normal serum, incubated with anti-Aβ and stained buffer for 30 min at room temperature to eliminate
with diaminobenzoic acid (vector ABC Elite kit, vector endogenous peroxidase activity. After 1 h of blocking
laboratories). Bright field light microscopy imaged with 5.0% serum (horse or goat), the sections were
brain areas from which stained amyloid areas were incubated overnight with primary antibodies. Primary
quantitated using image analysis (NIH Image software, antibody and dilutions were: glial fibrillary acidic
NIH, Washington, DC). protein (GFAP)-positive astrocytes (1:200 dilution,
2E1; BD Biosciences, San Jose, CA). The next day,
Brain amyloid β analysis sections were washed 3 times (5 min/wash) with 0.1%
Brain Aβ analysis was conducted as previously Triton X-100 and TBS buffer to remove excess primary
described for transgenic AD mice. [27,28] Briefly, antibody. Thereafter, primary antibodies were detected
animals were sacrificed and brain extracts were using horseradish peroxidase (HRP)-conjugated
homogenized (1:3 weight/volume of buffer) in mouse immunoglobulin G Vectastain ABC kit
buffer of 5 mol/L guanidine HCl in 50 mmol/L and DAB/substrate reagents (vector laboratories,
Tris-HCl, pH 7.6, 150 mmol/L NaCl, plus protease Burlingame, CA) according to the manufacturer’s
inhibitors (Sigma). Homogenates were diluted to instructions.
0.5 mol/L guanidine and centrifuged (200,000 g
for 20 min), and supernatant and pellet fractions Enzyme-linked immunosorbent assays for inflammatory
were collected. The pellet from the brain extract markers
procedure was sonicated in 6 mol/L guanidine and Brain hemispheres were weighted and homogenized
centrifuged at 200,000 g for 20 min at 4 °C, and with 4 volumes of phosphate buffered saline
the supernatant was diluted to 0.5 mol/L guanidine. buffer (125 mg/mL) containing complete protease
The two supernatants were combined, and Aβ (40) inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO,
and Aβ (42) (Aβ 1-40 and Aβ 1-42 , respectively) were USA). The supernatant was then collected, and total
determined using enzyme-linked immunosorbent protein was determined by the BCA method (Pierce
assays (ELISAs) kits specific for each peptide (IBL, Biotechnology, Rockford, IL, USA). Tumor necrosis
JP27718 and JP27711). ELISAs measured Aβ peptides factor (TNF)-α, interleukin (IL)-1β and IL-6 levels
by methods previously described. [29-33] Protein content were measured with a mouse TNF-α, IL-1β and IL-6
was determined by the Bradford method. ELISA kits (R and D Systems, Minneapolis, MN, USA).

Neuroimmunol Neuroinflammation | Volume 2 | Issue 1 | January 15, 2015 33