1. Home
  2. Education
  3. Best IHC Staining Practices

Methods & Controls

Staining Guide | Methods & Controls | Troubleshooting | Video Training | Download Guide

Methods

Manual Staining

Operator error can result in:
Weak or No staining no reagent or incorrect reagent application or a possible reagent mix-up.
Variable staining insufficient or uneven washing.

Tech Tip: It is important to never let pretreated tissue dry-out; always keep your slides in wash buffer in-between the steps of your manual staining procedure.

Automation/Instrumentation

Operator error can result in:
Weak or No Staining no reagent or incorrect reagent is applied, incorrect protocol set-up (mapping), and/or a possible reagent mix-up.

An instrument malfunction can result in:
Weak or No staining no reagent or incorrect reagent is applied.
Variable staining insufficient or uneven washing.

Tech Tip: There are 2 types of automated platforms:

“Open” systems that allow the use of any vendor’s detection, antibodies, buffers, and ancillaries.

“Closed” systems that require use of the manufacturer’s detection, buffers, and ancillaries; only antibodies from other vendors can be used with these instruments.

Controls

Positive control: tissue known to be immunoreactive to the primary antibody.

  • Verifies that reagents were applied properly and staining was performed correctly
  • Included every time a staining run is performed with a particular antibody
  • Guards against false negative staining

If a positive tissue control is negative reagent did not get applied, reagents were mixed-up, wrong or insufficient pretreatment, reagents were expired or stored improperly, and/or there could be a tissue fixation issue.

Tech Tip: A positive control can be any tissue type, normal or diseased, that demonstrates an antibody’s immunoreactivity; you do not have to use the same tissue type as the patient tissue.

Negative control: patient tissue where primary antibody is NOT applied.

  • Included every time a staining run is performed with a particular antibody
  • Guards against false positive staining

If a negative tissue control is positive endogenous (biotin, peroxidase, phosphatase) elements may be picking up chromogen or interacting non-specifically with components within the detection system.