Biology Reference
In-Depth Information
Table 3
Day 1: Incubation in primary antibodies
Immunostaining
Incubation buffer (IB) a
Blocking buffer (BB)
Incubation solution (IS)
Primary antibodies b
Calculations
0.5 ml (500 μl) for each
section x10
sections = 5,000 μl
i.e. total volume of IB
for 10 sections
is 5,000 μl
BB is a mixture of IB
and normal serum:
5,000 μl × 5% =250 μl
(required volume of
NGS c ):
i.e. 4,750 μl of IB plus
250 μl of NGS
Split prepared BB into two
10-ml tubes for each
immunostaining
Subtract volume required for
incubation of negative
control section: 2,500 μl-
500 μl = 2,000 μl
IS is a mixture of BB and primary
antibody
For each staining :
Total volume of incubation
solution is 2,000 μl:
500 μl for each
section × 4 sections = 2,000 μl
For each staining (1:200):
2,000 μl:200 = 10 μl of each
antibody:
I. (i) anti-CD3 10 μl
(ii) anti-CD4 10 μl
II. (i) anti-CD3 10 μl
(ii) anti-CD8-alpha 10 μl
I. CD3/CD4 For four brain sections
CD3
CD4
0.3% Triton-X 100 in
Wash buffer: 0.5 ml
for each section
NGS
2,000
μ
l of BB
Mouse monoclonal anti-CD3
(PC3/188A), sc-20047, 1:200
Rabbit polyclonal anti-CD4
(H-370) sc-7219, 1:200
10
μ
l anti-CD3
10
μ
l anti-CD4
II. CD3/CD8 For four brain sections
CD3
0.3% Triton-X 100 in
Wash buffer: 0.5 ml
for each section
NGS
2,000
μ
l of BB
Mouse monoclonal anti-CD3
(PC3/188A), sc-20047, 1:200
Rabbit polyclonal anti-CD8 alpha
(H-160) sc-7188, 1:200
no primary antibodies
10
μ
l anti-CD3
CD8-alpha
10
μ
l anti-CD8 alpha
Negative control
(2 sections)
0.3% Triton-X 100 in
Wash buffer: 0.5 ml
for each section
NGS
no primary antibodies
a See recipe in Subheading 2.7
b Santa Cruz Biotechnology, Inc., CA, USA
c NGS normal goat serum
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