Light Microscopic Assessment - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 461

Taken
without
perfusion-fixation

Taken
after
perfusion-fixation

Fresh, non-fixed
specimen

Fixed specimen

Freezed
in
OCT compound

Embeded
In
paraffin

Sectioned with a cryotome
Dried
Fixed with acetone or ethanol

Sectioned with a microtome
Deparaffinized with xylene
Rehydrated

Incubated with 3% H2O2

Washed with PBS

Incubated with blocking solution

Incubated with a primary antibody

ABC method, LSAB method, or labeled polymer method

Fig. 3. Flowchart of immunostaining.

In ABC method, after being washed with PBS, the section is
incubated with a HRP labeled secondary antibody at room
temperature.
In LSAB method, after being washed with PBS, the section is
incubated with a secondary antibody at room temperature and
then with HRP-conjugated streptavidin.
In labeled polymer method, after being washed with PBS, the
section is incubated with a simple stain PO solution at room tem-
perature for 30 min.
And in any methods, immunoreactive antigen is visualized by
incubation with DAB.