Synthetic Antigen Controls for Immunohistochemistry

BSA antigen gels are well-characterized reproducible IHC standards that can supplement existing controls. This protocol uses standard histology and IHC reagents and methods to make reproducible standards of known composition. These controls provide the opportunity for different labs to calibrate and standardize IHC assays for many diagnostically relevant assays.

It is important to find a suitable solvent to dissolve the peptide or protein. It can be tricky to mix antigen and formaldehyde solutions quickly without introducing bubbles. To begin, prepare 20 milliliters of a 25%weight by volume BSA solution by mixing five grams of BSA powder in 14 milliliters of PBS, in a 50 milliliter conical tube until evenly distributed.

Vortex the solution to dissolve the BSA powder. Keep the solution overnight at four degrees Celsius for complete dissolution. The following day, adjust the final volume to 20 milliliters with PBS.

Similarly, prepare 20 milliliters of a 31.3%weight by volume BSA solution by mixing 6.26 grams of BSA powder in 13 milliliters of PBS. After overnight incubation for complete dissolution, adjust the final volume to 20 milliliters with PBS. To test that the BSA formaldehyde mixture forms a gel, preheat a heat block to 85 degrees Celsius.

Then mix 700 microliters of the 25%BSA solution with 700 microliters of 37%formaldehyde. Mix well by pipetting up and down five times within five to 10 seconds without creating air bubbles. Immediately after mixing, place the closed micro centrifuge tube in the heat block preheated to 85 degrees Celsius.

After 10 minutes, remove the tube from the heat block. Allow it to cool and confirm that the gel has formed as expected. Prepare and clearly label eight 1.5 milliliter micro centrifuge tubes.

Then prepare a 5x peptide stock solution, add a 12.5 millimolar concentration by resuspending the entire mass of the liberalized peptide in 60 microliters of the appropriate solvent. Observe the solution to ensure that the peptide is completely dissolved. Then add an additional volume of solvent as necessary to make a 12.5 millimolar solution according to the peptides molecular weight, mass, and purity.

In this example, the 5x peptide stock has a final volume of 626.9 microliters. Other peptide samples will require a different final volume. Next, prepare 150 microliters of a 1x peptide stock solution add a 2.5 millimolar concentration by diluting 30 microliters of the 5x peptide stock into 120 microliters of the solvent.

Vortex the solution for five seconds and centrifuge at room temperature. Next, prepare 700 microliters of a 0.5 millimolar peptide BSA solution by diluting 140 microliters of the 1x peptide stock into 560 microliters of the 31.3%BSA solution. After vortexing, centrifuge the solution at room temperature.

Similarly, prepare four successive 10x serial dilution of the peptide BSA stock by adding 70 microliters of the peptide BSA solution to 630 microliters of the 25%BSA solution. To prepare the BSA peptide gels, add 37%formaldehyde to the peptide BSA dilutions in a one to one ratio working one sample at a time. Mix well by pipetting up and down five times within five to 10 seconds without creating air bubbles.

After mixing, place the closed micro centrifuge tube in a heat block at 85 degrees Celsius for 10 minutes. After all the peptide BSA and formaldehyde solutions have been prepared and heated, allow the gels to cool on the benchtop for five to 10 minutes. Next, using a clean, flexible disposable laboratory spatula, remove the gel sample from the micro centrifuge tube in one piece.

And place it in a sealed container containing at least 15 milliliters of neutral buffered formalin, using a separate container for each sample. Alternatively, using a new single edge razor blade, cut off the bottom of the micro centrifuge tube and push the gel out from the bottom with air or a suitable probe. Using a clean single edged razor trim the gel cone into cylindrical discs approximately five millimeters thick.

After wrapping the discs in a biopsy wrap, place one larger gel disc into one cassette and the remaining gel discs together into a second cassette for use in tissue microarray construction. Before processing, place the cassette gels in at least 15 milliliters of 10%neutral buffered formalin per gel, using a separate container for each sample. Next, process the gels in an automated histology tissue processor following a large tissue schedule with pressure and vacuum.

When the sample processing is complete, remove the cassettes from the tissue processor and move them to the paraffin embedding center. After unwrapping the gels from the biopsy wrap, embed the gels in paraffin or each sample embed one disc of gel in a small 15 by 15 millimeter mold. And the remaining gel discs together in a second larger mold.

For each peptide dilution series, create two glass slides containing a total of six separate sections. One section from each of the five dilution series samples and one section from the BSA only negative control sample. After sectioning and drying the slides, stain the two slides prepared for each peptide with the desired primary antibody.

According to standard Immunohistochemistry protocols expect to see a relatively uniform signal within each gel section. With the different gel samples showing a range of signal intensity corresponding to the peptide dilutions. For the BSA gel tissue microarray construction, cut four micrometer thick sections of the tissue microarray and stain with a rabbit monoclonal antibody according to laboratory standard protocols.

Assess the resulting stain intensity qualitatively by inspection or quantitatively buy digital image scanning and analysis. After reacting with the appropriate antibodies, negative control BSA only gel samples showed minimal signal. The signal intensity in an individual gel sample is relatively uniform and increases with increasing antigen concentrations.

More than 10%of MDA-MB-175 cells show faint and in completely circumferential membranous staining. In contrast, more than 10%of the SKBR-3 cells, show intense completely circumferential membranous staining Work quickly once the formaldehyde has been added because the solutions will begin to gel even at room temperature. We use this method to make IHC controls when testing new antibodies to give us confidence that the assay performed as expected, even when the test antibodies show no signal.

Tissue or cell line controls are inherently variable. Synthetic antigen controls allow us to measure the effect of changing specific ISD reaction conditions more precisely.