Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 113

3.6 Analysis
of Brain Tissues

We routinely analyze A deposition and neurogenesis in the AAV-

injected hippocampal region of AD mouse models. For BrdU stain-
ing, a special preparation is necessary for nucleic acid staining.

ʲ

3.6.1 Histological
Analysis of Gene
Expression, Neurogenesis,
and Amyloid Pathology
with Immunofl uorescence

Immunohistochemistry for BrdU
We have used this protocol for free-fl oating mouse brain sections,
sectioned at 30 µm. Binding of the antibody to BrdU is
achieved by denaturing the DNA through exposure of tissue
to heat and acid, followed by a traditional immunohistochem-
istry protocol.

3.6.2 Analysis
of Neurogenesis

Day 1:

1. Rinse the sections in PBS 3 × 5 min.
2. Antigen retrieval: Incubate the sections in 50 % for-
mamide/50 % 2× saline-sodium citrate (SSC) solution at 65 °C
for 2 h on a shaker. Be sure to make the formamide/2× SSC
solution in a chemical fume hood. Heat the solution to 65 °C,
and then add to the sections.
3. Rinse the sections with 2× SSC, pH 7.4 at room temperature
for 10 min.
4. Incubate in 2 M HCl at 37 °C, 30 min.
5. To neutralize the acid, rinse the sections in 0.1 M borate buffer
at room temperature for 10 min.
6. Wash in PBS 3× 5 min.
7. The following steps are a traditional immunohistochemistry
with anti-BrdU antibody and may be adjusted to fi t your lab's
protocols.

DAB staining for A

For diaminobenzidine (DAB) staining of A

ʲ 3.6.3 Analysis of Amyloid

Pathology

ʲ , we use a 2-day pro-

peptide antibody from
Invitrogen. DAB is a hazardous chemical and is harmful if
swallowed, inhaled, or placed in contact with the skin. It is a
suspected carcinogen. Appropriate precautions should be used
for handling and disposal of DAB.
Thiofl avin-S Staining
Thiofl avin-S is a sulfur-containing compound that binds to amy-
loid fi brils and has been suggested to be a more specifi c stain
than Congo red or Thiofl avin T [ 107 ]. For TS staining, the
following protocol may be used, based on the protocol of
Selkoe [ 108 ].

tocol and a rabbit polyclonal A ʲ