TITLE: Dual antibody immunohistochemistry staining
AUTHORS: Angelina Spence, Mako Goldston, Kausalia Vijayaragavan, Marc Bosse, Sean Bendall, Mike Angelo

[DESCRIPTION]
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]
SECTION: Protocol IO references
1. 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
SECTION: Slide preparation
2. Slides are baked, deparaffinized and processed for antigen retrieval and washed in PBS wash buffer as described in the IHC staining protocol.
SECTION: Slide preparation
4. 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.
SECTION: Slide preparation
3. The slides are then placed in an immunostain humid chamber.
SECTION: Slide preparation
5. After blocking, the slides are briefly rinsed with PBS wash buffer and mounted on the Sequenza assembly  (ref. Sequenza protocol).
SECTION: Slide preparation
6. Add 2 mL of PBS wash buffer for each slide and let the entire volume of buffer flow through.
SECTION: Slide preparation
7. Add 200 µL of blocking buffer (ref. MIBI IHC solutions) and incubate 1h.
SECTION: Primary antibody staining titration
7.1. 
 

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.
 

SECTION: Primary antibody staining titration
8. 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. 
SECTION: Primary antibody staining titration
9. 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.
SECTION: Double staining
10. 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.
SECTION: Double staining
11. After blocking, add 200 µL antibody diluent and let the entire volume of buffer flow through.
SECTION: Double staining
12. Add 100 µL of the diluted antibodies per slide and incubate at 4°C overnight.
SECTION: Double staining
13. The next day, add 2 mL of PBS wash buffer and let the entire volume of buffer flow through.
SECTION: Double staining
15. 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).
SECTION: Double staining
16. Incubate for 10 min at room temperature.
SECTION: Double staining
17. Add 2 mL of PBS wash buffer and let the entire volume of buffer flow through.
SECTION: Double staining
14. Bring all the reagents for double IHC staining to room temperature. 
SECTION: Development
18. 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.
 

SECTION: Development
19. 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.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 . 
 

SECTION: Development
20. 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). 
SECTION: Development
21. Rinse in water prior to the peroxidase development.
SECTION: Development
22. Prepare the peroxidase substrate working solution by adding the components in the recommended buffer as instructed by the manufacturer.
SECTION: Development
23. 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.       
SECTION: Development
24. Stop the development by removing the excess and transfer in water.
SECTION: Development
25. 
 

SECTION: Development
26. Dehydrate the tissue in 1x ethanol 70%, 1x ethanol 80%, 2x ethanol 95%, 2x ethanol 100%.
SECTION: Development
27. Coverslip in a non-aqueous mounting media such as VectaMount (Vector Laboratories, H-5000).


 

SECTION: Development
28. Let the slides dry completely before visualization.