This protocol may be used to perform immunohistochemistry on either paraffin embedded or frozen sections.


Category
Protocol
Record History
Added on June 15, 2010 at 10:19 AM by Lindner, Jill
Modified on July 1, 2010 at 3:20 PM by Lindner, Jill
Shared with (contributions)
Public: Shared Collection

Reagents, supplies, and equipment:

  • PBS
  • PBS-T (0.1% Tween 20 in PBS)
  • xylene
  • 100%, 95%, 70% ethanol in H2O
  • antigen retrieval buffer (for 1 L citric sodium 2.94 g, pH 6 adjusted with 10% citric acid)
  • 30% hydrogen peroxide
  • Dako Cytomation pen
  • slide staining jars

Procedure: (de-paraffining, for paraffin embedded sections)

Sections are treated in the rectangular slide staining jars under the hood:

  1. 3 x 5 min in xylene
  2. 2 x 5 min in 100% ethanol
  3. 1 x 5 min in 95% ethanol
  4. 1 x 5 min in 70% ethanol
  5. 1 x 5 min in tap water

Procedure: (section cleaning, for frozen sections)

  1. Wash 2 x 5 min in tap water
  2. Wash 1 x 5 min in PBS

Endogenous peroxidase activity inhibition (if peroxidase is to be used) - To inhibit endogenous peroxidase activity, sections are incubated in 3% H2O2 for 20 min at RT.

Antigen retrieval (optional, recommended for nuclear antigens)

  1. Heat sections to 90°C (do not boil).power level 1.
  2. Cool sections at RT for 20 - 30 min.

Procedure: - Immunolabeling

  1. Circumscribe individual sections with pap pen.
  2. In order to block non-specific binding, incubate sections in 3% BSA in PBS-T for 30 min at RT.
  3. Incubate sections with the primary antibody diluted at the proper concentration in 3% BSA in PBS-T O/N at 4°C.
  4. Wash sections 3 x 5 minutes in PBS-T
  5. Employ one of the following secondary antibody staining techniques:
  • The secondary antibody is directly linked to the enzyme.
    a.  Incubate the sections with secondary antibody for 1 hr at RT.
    b.  Wash 2 times with the appropriate buffer (PBS for HRP or Tris-HCl 10 mM pH 8.6 for alkaline phosphatase).
    c.  Incubate with the proper substrate and control the staining under the microscope.
    d.  Wash 2 x 5 min in H2O, then mount with mouthing solution.
    OR
  • The secondary antibody is linked to biotin.
    a.  Incubate the sections with the secondary antibody coupled to biotin for 1h at RT.
    b.  Wash 2 times in PBS.
    c.  Incubate with the avidin- AP or HRP complex for 30 min at RT (Prepare complex at least 30 min in advance.).
    d.  Wash 2 times with the appropriate buffer.
    e.  Incubate with the proper substrate and control the staining under the microscope.
    f.  Wash 2 x 5 min in H2O then mount with the mounting solution.

Written by Bertrand Blondeau 11/17/2003

Revised 1/13/2004