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National Human Neural Stem Cell Resource
Philip H. Schwartz, PhD, Director
Children's Hospital of Orange County
455 South Main Street
Orange, CA 92868-3874
Phone: 714-516-4310
800-418-1961
1. Remove the brain, with cerebellum and brainstem intact;
cut mid-sagittally and separate the cerebral hemispheres.
2. Fix the left 1/2 of brain in 4% neutral buffered formaldehyde
(10% formalin). When fixation is complete, drain away excess
fixative, package brain in heat sealed plastic bag (x2) or
airtight plastic container, and send entire brain to repository
(see notes).
3. Remove the brainstem and cerebellum from the other half
of the brain by sectioning transversely through the cerebral
peduncles at the most anterior level of the colliculi. This
gives "Section A".
4. Section the cerebellum, parasagittally, just lateral to
the most lateral extent of the brainstem and again 1 cm lateral
to this. This second cut gives "Section B".
5. Section the remaining hemisphere, after placing the medial
surface down, coronally at 1cm intervals from the frontal
to the occipital poles. The coronal section just posterior
to the most anterior extent of the temporal lobes will be
"Section D". The section 2cm posterior to that (or
3cm posterior to the most anterior extent of the temporal
lobe) will be "Section C".
6. Dissect out from the appropriate sections (see above and
Figures linked to below):
- Substantia nigra (from Section
A).
- Cerebellar cortex (from Section
B).
- Hippocampus,
- Centrum semiovale, and
- Cortex (from Section C).
- Periventricular zone from the head of the caudate nucleus,
and
- Caudate nucleus (from Section
D).
7. Place brain pieces in separate petri dishes with sterile
cell culture medium containing antibiotics. Rinse the tissue
three times with medium. Making the cut orthogonal to the
axis of the structure, trim off a 2mm thick slab from each
tissue piece and place them in individual vials containing
10mL fresh buffered 4% paraformaldehyde. Then replace the
medium for the remaining tissue with medium containing DMSO
(approx. 1.5 mL/100 mg tissue). Using sterile scalpel blades,
mince brain into pieces that are less than 1 mm in any dimension.
Transfer semi-equivalent aliquots of minced tissue/medium
(approx. 1.5 mL) into Nalgene cryovials and freeze slowly
in isopropanol freezing containers. Store at -70°C for
1 - 7 days and then ship to the NHNSCR on dry ice by FEDEX.
Ship the paraformaldehyde vials, on ice-packs, by FEDEX to
the NHNSCR, immediately.
8. Prepare remainder of fresh brain and other tissues according
to standard protocol, below.
Option 1: Rinse tissues three times with sterile cell culture
medium containing antibiotics. Place the tissues in sterile
medium in sterile falcon tubes then ship all vials and tubes,
with ice packs, immediately to the NHNSCR by FEDEX.
Option 2: Place entire brain in sealed plastic bag (x2), place
bag on wet ice in insulated container and FEDEX or courier
to NHNSCR.
Notes: 1) All media and disposable supplies will be provided
by the NHNSCR. The NHNSCR will also pay all courier and/or
FEDEX charges.
2) The protocol may differ substantially for very small or
misshapen brains. Contact Dr. Schwartz to discuss this on
a case-by-case basis.
3) The repository to which the excess fixed and frozen tissues
will be sent will be decided on a case-by-case basis by Dr.
Schwartz.
4) Tissues, other than brain, to be procured will depend on
the case and will be decided on a case-by-case basis in consultation
with Dr. Schwartz.
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