Biology Reference
In-Depth Information
13. Secondary antibodies.
14. Vectashield mounting media.
15. Cover slip. It is critical to use coverslips suitable for fl uores-
cence (for example, Fisher scientifi c, Fisherfi nest Premium
cover glass, #12548B).
16. Gooseneck lamp.
17. Ice container.
18. 3D rotator.
19. Aluminum foil.
Protocol
During the immunostaining procedure use double-distilled water
for buffers and rinse plastic dishes well before and after use.
I. Day 1
(Table
3
)
1. Using paint brush, transfer the desired brain sections into
24-well plates loaded with 1 ml of Wash buffer in each well.
2. Rinse sections three times, 10 min each in Wash buffer on 3D
rotator.
3. Prepare blocking buffer (see calculation sample in Table
3
):
(a) Take prepared Incubation buffer (0.3% Triton-X 100 in
Wash buffer) and add 5% NGS.
(b) Gently mix to avoid bubbling.
4. Place 0.5 ml of blocking buffer into wells proposed for nega-
tive control sections.
5. Divide remaining blocking buffer into two 10-ml plastic
tubes for further incubation with: (a) CD3/CD4 and (b)
CD3/CD8 primary antibodies.
6. Briefl y (~5 s) centrifuge tubes with primary antibodies prior to
opening (Fisher Scientifi c Minicentrifuge, cat.# 05-090-100,
maximum speed/force 6600 rpm/2200 ยด
g
can be used).
7. To prepare an incubation solution containing the primary anti-
bodies, add each into each of 10-ml plastic tubes containing
the blocking buffer (see step 5). Our primary antibodies were
diluted 1:200 (see Table
3
and Fig.
4
). Primary antibody con-
centrations can vary; it is recommended that the affi nity of the
antibody will be tested with a series of dilutions.
8. Gently mix and load 0.5 ml of incubation solution containing
the primary antibody into each well.
9. Transfer sections into 24-well plates containing loaded incuba-
tion solution.
10. Incubate the negative control brain sections in a separate
24-well plate loaded with blocking buffer (step 4 in protocol).
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