Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 614

(c) Aspirate solution from 1.5-ml tube containing secondary
antibodies into 1-ml syringe and fi lter it through a 0.20-

m syringe fi lter into 10-ml tube with blocking buffer.
16. Repeat steps 7-14 from immunofl uorescence protocol, Day 2.
Decrease the incubation time with secondary antibodies to
1 h. Use only clear coverslips. Do not allow slides to dry.

μ

Note : The secondary antibody should be against a species that
the primary antibody is raised. For example, if the primary anti-
body is raised in mouse, an anti-mouse secondary antibody should
be used. If it is raised in rabbit, an anti-rabbit secondary antibody
should be used.

We summarize a few the most common problems with fl uorescent
and near-infrared dye labeling in Table 5 .

2.6. Troubleshooting

Table 5
Troubleshooting table

Problem Possible reason Action

A. Fluorescent dye labeling
CD3/CD4/CD8 non-
specifi c staining with
and/or high background

Non-specifi c binding of
primary or secondary
antibody
Incubation with primary
CD3/CD4/CD8
antibody was too long
Not enough concentration
or not suffi cient block
reagent

Increase the number and time of
washes in between steps

Reduce incubation time with
primary antibody

Increase concentration and/or
additionally incubate sections with
blocking reagent prior to main
procedure;
Make sure that blocking serum is
from the same species as the host
of the secondary antibody

B. Near-infrared dye labeling
Visible artifacts in color
distribution—brighter
artifi cial colors and cracked
tissue at the brain edges

Slides were left to dry Make sure that slides are covered

during incubation
Use agitation for all antibody
incubations
Do not let slides dry out and keep
moist at all times during the
staining procedure; cover with
clear Vectashield coverslips
Adjust incubation time
LI-COR scanner allows adjustment
signal intensities over a wide range;
however, it is critical to keep the
scanning parameters consistent
between scanned sections

Too bright, saturated, or poor
infrared signal intensity
on the section scans

Either too long or too
short time of incubation
with secondary
antibodies