Dual antibody immunohistochemistry staining
Marc Bosse, Sean Bendall, Mike Angelo, Angelina Spence, Mako Goldston, Kausalia Vijayaragavan
Abstract
This protocol provides guidelines to perform dual antibody immunohistochemistry ( IHC) on standard FFPE tissue in the Sean C. Bendall and Michael R. Angelo labs. The staining procedure is principally performed to validate the co-occurrence of two targets observed in Multiplex Ion Beam Imaging Time of Flight (MIBI_TOF) analysis.
Steps
Protocol IO references
This protocol refers to detailed procedures found in these respective Protocol IO:
- IHC staining dx.doi.org/10.17504/protocols.io.bf6ajrae
- Sequenza dx.doi.org/10.17504/protocols.io.bmc6k2ze
- MIBI and IHC solutions dx.doi.org/10.17504/protocols.io.bmc6k2ze
Slide preparation
Slides are baked, deparaffinized and processed for antigen retrieval and washed in PBS wash buffer as described in the IHC staining protocol.
The slides are then placed in an immunostain humid chamber.
To block endogenous peroxidase and alkaline phosphatase, the section of each slide is covered with 3-4 drops of Bloxall (Vector Laboratories, SP-6000-100), and incubated for 30 min at room temperature.
After blocking, the slides are briefly rinsed with PBS wash buffer and mounted on the Sequenza assembly (ref. Sequenza protocol).
Add 2 mL of PBS wash buffer for each slide and let the entire volume of buffer flow through.
Add 200 µL of blocking buffer (ref. MIBI IHC solutions) and incubate 1h.
Primary antibody staining titration
Perform an antibody titration for each antigen target. The recommended starting titers would be 1 µg/mL and 0.25 µg/mL with known antibody concentration or use the provider recommended dilution titer and 4x less (e.g. 1:400 and 1:1600). From the results of the first titration, iterate with a second titration to narrow it down. This initial titration can be done using the HRP and DAB detection. The DAB revelation time should be between 30s to 1 min, no less.
Once the initial titer is determined, perform a single color stain for each target with each substrate. Choose the color substrate that gives the best sensitivity for a putative target. In contrast to DAB, some substrates have a wide range of development time which gives more flexibility to increase sensitivity. For example, the alkaline phosphatase substrate Vector blue (SK-5300) has an optimal revelation time of 20-30 min but can be extended from 2 to 4 hours.
Once the secondary-enzyme conjugate, the color substrate, and the development time for each antigen target have been determined, proceed to perform the double staining.
Double staining
Dilute the antibody with the lowest titer first (Antibody 1) in antibody diluent buffer. Then dilute the second antibody (Antibody 2) using the solution of Antibody 1 with the lower titer.
After blocking, add 200 µL antibody diluent and let the entire volume of buffer flow through.
Add 100 µL of the diluted antibodies per slide and incubate at 4°C overnight.
The next day, add 2 mL of PBS wash buffer and let the entire volume of buffer flow through.
Bring all the reagents for double IHC staining to room temperature.
For each slide, add 3 drops of the secondary antibody-enzyme mix corresponding the desired color for each target (ImpressDuet, MP-7714 or MP-7724, Vector Laboratories).
Incubate for 10 min at room temperature.
Add 2 mL of PBS wash buffer and let the entire volume of buffer flow through.
Development
Start revealing with the alkaline phosphatase substrate first. Prepare the alkaline phosphatase substrate working solution by adding the components in the recommended buffer as instructed by the manufacturer.
Dismount the Sequenza assembly and put the slides on a immunostain humid chamber. Add immediately an excess of substrate working solution to cover the tissue section. .
Stop the development by removing the excess and incubate for 5 min in recommended buffer (100 mM Tris-HCl pH 8.5, Tween 0.1% or PBS).
Rinse in water prior to the peroxidase development.
Prepare the peroxidase substrate working solution by adding the components in the recommended buffer as instructed by the manufacturer.
Put the slides back into the immunostain humid chamber. Immediately add an excess of the peroxidase substrate working solution to cover the tissue section and develop for 40s.
Stop the development by removing the excess and transfer in water.
Dehydrate the tissue in 1x ethanol 70%, 1x ethanol 80%, 2x ethanol 95%, 2x ethanol 100%.
Coverslip in a non-aqueous mounting media such as VectaMount (Vector Laboratories, H-5000).
Let the slides dry completely before visualization.
