Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 612

Table 4
Day 2: incubation in secondary antibodies

Immunostaining Incubation buffer (IB) Blocking buffer (BB) Incubation solution (IS) Secondary antibodies

A. Calculations

Total IB volume for 10
sections is 5,000

5,000 μ l × 5% = 250 μ l; i.e. 2,500 μ l of IB, 12.5 μ l AF 2,500 l of each AF

568 and AF 488 conjugated
antibodies
2,500

μ l:200 = 12.5 μ

μ l 4,750 μ l IB plus 250 μ l 568, 12.5 μ l AF 488

of NGS
split BB into two 10 ml tubes
each contain 2,500

2,500 μ l BB, 10 μ l CF 680, l of each CF 680

and CF 770 conjugated antibodies

μ l:250 = 10 μ

μ l 10 μ l CF 770

B. Fluorescent dye
labeling
I. CD3/CD4:
2 sections

0.5 ml IB for each
section

NGS a 2,500 μl of IB 12.5 μl AF

568 12.5 μl AF 488

Alexa Fluor 568 goat anti-mouse
IgG (H + L), Invitrogen
Corporation, Carlsbad, CA, USA;
1:200
Alexa Fluor 488 F(ab¢) 2 fragment of
goat anti-rabbit IgG (H + L),
Invitrogen Corporation, Carlsbad,
CA, USA; 1:200

II. CD3/CD8:
2 sections
III. Negative control:
1 section

0.5 ml IB for each
section
0.5 ml IB for each
section

NGS 2,500 μl of IB 12.5 μl AF

568 12.5 μl AF 488
2,500 μl of IB 12.5 μl AF
568 12.5 μl AF 488

NGS

B. Near-infrared
dye labeling
I. CD3/CD4:
2 sections

0.5 ml IB for each
section

NGS 2,500 μ l BB 10 μ l CF 680 CF 680 goat anti-mouse IgG,

Biotium, Inc., Hayward, CA USA;
1:250
CF 770 goat anti-rabbit IgG,
Biotium, Inc., Hayward, CA USA
1:250

10 μ l CF 770

II. CD3/CD8:
2 sections

0.5 ml IB for each
section

NGS 2,500 μ l BB 10 μ l CF 680

10 μ l CF 770

III. Negative control:
1 section

0.5 ml IB for each
section

NGS 2,500 μ l BB 10 μ l CF 680

10 μ l CF 770

a NGS normal goat serum