Materials – 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. That 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 5000 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.
Product Arrival –
- Store in the refrigerator, not freezer.
- Before using for the first time, centrifuge the bottle for 3 – 5 seconds on the lowest setting of a centrifuge. A mini tabletop centrifuge can be ideal, as illustrated in the figure (above). The purpose of the flash spin is to sediment the reagent to the bottom of the tube. (During shipment, the bottle is invariably inverted. Some of the reagent may be trapped underneath the lid. The flash spin will collect the reagent in the bottom of the vial).
- Repeat the flash spin any time reagent collects underneath the lid. This can occur if the bottle is inverted or by evaporation and condensation, during storage. After centrifugation, please remember to then vortex mix the tube before using.
Mixing – 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. To cause effective mixing, hold the tube at an angle on the rubber vortex mixing head.
Avoid the wax ribbon – It is important to avoid dispensing on top of wax. Sometimes, there is a thin layer of wax even though it is not visually apparent. To ensure consistency, wipe the area of the slide (adjacent to the tissue section) with a Kimwipe, gauze, or similar, where you intend to apply the IHControls™, as shown in the figure below.
After dispensing 1 microliter of the IHControlTM, allow the droplet to dry. If you are baking the slide, then that will harden the droplet. If you are not baking the slide, then let the droplet dry for at least 15 minutes at room temperature. As the spot dries and hardens, it adheres to the glass.
IHControls™ Post-fixation – 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 IHControlsTM 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.