IHConctrols® are simple to use and can be easily and cost-effectively purchased off-the-shelf to provide consistent results. Finally, labs can eliminate the time-consuming task of producing inconsistent, non-standardized homemade controls.
IHControls are intended to be applied to a slide bearing a formalin-fixed, paraffin-embedded tissue section.
Apply the control after the section is mounted on the slide. This allows you to position the control adjacent, but not too far away. A pipetter capable of dispensing 1 microliter is needed. Dispense accuracy is not important.
There are approximately 5,000 microbeads per microliter. With larger volumes, more microbeads are dispensed; with lower volumes, fewer microbeads are dispensed. Regardless, there will be more than enough microbeads on which to judge stain intensity.
The glass microbeads suspended within the IHControls are heavier than biological cells. Therefore, they sediment quickly, even without centrifugation. For that reason, mixing the fluid in the vial prior to dispensing is important. Once the vial is vortex mixed, dispense the microliter within 60 seconds so as to ensure an even distribution of microbeads in the vial. For effective mixing, hold the tube at an angle on the rubber vortex mixing head.
For xylene de-paraffinization, no post-fixation is required. The spot of IHControls adheres to and retains the microbeads on the microscope slide perfectly well during de-paraffinization and antigen retrieval.
For de-paraffinization and antigen retrieval performed on the Dako PT Link, using an alkaline buffer, an additional 15-minute post-fixation step is needed to further harden the matrix holding the microbeads onto the slide. This extra step is sometimes needed because the detergents used in the PT Link de-paraffinization solutions disrupts the dried IHControls spot.
This additional fixation is called “post-fixation” because the HER-2, ER, and PR peptide antigens are already formaldehyde-fixed during manufacture. The post-fixation step cross-links components in the liquid matrix in which the microbeads are suspended.
The post-fixation step involves placing a small droplet of neutral buffered formalin on top of the dried on-slide IHControls spot. Incubate for 15 minutes at room temperature. Then, rinse off the formalin droplet with 2 squirts of water from a transfer pipette, let it dry, and it is ready to use.