Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 613

(b) Add suffi cient volume of 5% normal goat serum (see
Table 4 ).
(c) Gently mix Incubation buffer with serum but avoid
bubbling.
4. Divide blocking buffer into two 10-ml plastic tubes proposed
for further incubation with immunofl uorescent and infrared
labeled secondary antibodies (Fig. 4 ).
5. Before opening, briefl y (~5 s) centrifuge tubes containing
secondary antibodies at high speed (~6,600 rpm) to remove
aggregates.
6. To prepare an incubation solution for immunofl uorescence:
(a) Transfer about 1 ml from 10-ml tube containing blocking
buffer into 1.5-ml tube.
(b) Add calculated volumes of Alexa Fluor 568 and Alexa
Fluor 488 conjugated secondary antibodies into 1.5-ml
tube with blocking buffer, i.e. 1.5-ml tube should get both
Alexa Fluor 568 and Alexa Fluor 488 conjugated second-
ary antibodies.
(c) Aspirate solution from 1.5-ml tube containing secondary
antibodies into 1-ml syringe and fi lter it through a 0.20- μ m

syringe fi lter into 10-ml tube with blocking buffer.
7. Gently mix. Using a separate 24-well plate, load 0.5 ml of
incubation solution into each well.
8. Transfer brain sections into wells containing the incubation
solution.
9. To avoid contamination, incubate the negative control sections
in a separate 24-well plate loaded with the same incubation
solution used for staining sections.
10. Cover the 24-well plates with aluminum foil and incubate
staining and negative control sections in incubation solution
for 2 h at room temperature on 3D rotator.
11. Rinse sections three times, 10 min each in Wash buffer on 3D rotator.
12. Carefully mount sections on glass slides with mounting media.
13. Cover with clear or DAPI-treated (4 ¢ ,6-diamidino-2-phenylindole)
coverslips.
14. Store slides in the dark at 4°C.
15. To prepare an incubation solution for infrared immunostaining:
After completing steps 1-5 from protocol, Day 2:
(a) Transfer about 1 ml from 10-ml tube containing blocking
buffer into 1.5-ml tube
(b) Add calculated volume (see Table 4 ) of GF 680 and GF 770
conjugated secondary antibodies into 1.5-ml tubes contain-
ing the incubation buffer, i.e. 1.5-ml tubes should contain
both GF 680 and GF 770 conjugated antibodies (Fig. 4 ).