Biology Reference
In-Depth Information
between the sternum and the shoulders and insert a 23 gauge
needle (mounted on a 1 mL syringe) in the jugular vein, which
should be clearly visible under the neck skin. If you prick the
vein correctly, the blood will flow into the hub of the needle.
Keep the syringe firm and draw the blood. Once you have
obtained the desired amount of blood, slowly pull back the
needle and apply a slight amount of pressure to the puncture
site. Usually 100-200
L of blood is sufficient to measure by
ELISA the concentration of EPO or S100E in the serum of the
AAV injected rats.
37. The ACF can be also collected punching the anterior chamber
with a 30 gauge needle mounted on a 1 mL syringe. After
punching a hole in the anterior chamber the ACF flows out
from the puncture site and can be collected using a 20
μ
L
pipette. However, using this method it is possible that some
tear fluid is collected together with the ACF.
38. The eye can be also oriented using a catgut suture. Follow step 1
described in Subheading
3.6.4
. Then, pass the catgut
superficially through the temporal conjunctiva. Fix the catgut
making a double knot and finally cut the catgut in excess using
scissors. The suture can be performed under a dissecting micro-
scope, if required.
39. During eye cryosectioning (see Subheading
3.8.1
) the lens
may tend to fold on the retina wasting the section. To avoid
this, it is suggested to embed the optic cup rather than the
whole eye by removing the cornea and the lens. To do this,
dissect the eye under a dissecting microscope. Briefly, grasp the
cornea using tweezers, cut away the cornea using Vannas scis-
sors and finally remove the lens using tweezers.
40. In most cases the cauterized part of the sclera is not visible to
the naked eye. Therefore, to embed the eye in the oriented
way it is necessary: (1) to observe it under a dissecting micro-
scope; (2) to position it in the desired orientation using twee-
zers; and (3) to transfer it into the mold preserving the desired
orientation.
41. The intramuscular injection of AAV vectors in rats is quite a
reproducible method therefore the levels of EPO and S100E
in the serum of injected rats should not vary widely. The pro-
tein (EPO and S100E) concentrations obtained following AAV
delivery should be included in the EPO standard curve range
if assaying 10-50
μ
L of serum by ELISA.
42. Unlike intramuscular injections, the ACF levels of EPO and
S100E proteins are highly variable due to the inherent vari-
ability of the subretinal injection as well as of the ACF collec-
tion procedure. However, 0.1-5
μ
L of ACF by ELISA should
allow measuring EPO and S100E concentrations included in
μ
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