TITLE: Staining protocol for Imaging Mass Cytometry
AUTHORS: Vijayakumar R Kakade, Tifanny Budiman, Lloyd Cantley

[DESCRIPTION]
In Imaging Mass Cytometry (IMC), a high-resolution laser is combined with a mass cytometer that permits mass spectrometry-based, spatially reserved high-dimensional analysis of intact formalin-fixed paraffin-embedded (FFPE) tissues. The protocol summarizes the staining procedure for IMC using a cocktail of heavy metal conjugated primary antibodies identifying specific antigens. 

[GUIDELINES]
Protocol Quality Control metrics
Antibody validation: All primary antibodies included in the panel are validated either by the vendor or internal validation. Strategies considered for antibody validation include: 1) knockout/knockdown of gene of interest, 2) independent antibody verification, 3) morphological validation by performing IF using single common protocol on human kidney and positive control tissue (lymph node, tumors etc), 4) morphological validation by a renal pathologist blinded to the antibody used and 5) counterstaining for additional markers of the same cell type.  
Aperio scanned image of PAS-stained section of the entire biopsy is evaluated for tissue morphology and integrity. 
Use 5 µm thick sections for staining. For better results, stain and ablate the tissue within 2 weeks of sectioning. 
Entire biopsy is ablated with increments in approximately 3 mm2 area to complete ablation of the cortical and medullary regions. Regions with artifactual section damage are excluded. 
Each IMC analysis includes a section from a reference kidney, both cortex and medulla (quality control), on the same slide as an internal standard. 
IMC is performed on the Hyperion imaging system. Prior to ablation of tissue, the machine undergoes routine tuning and calibration for mass spectrometric detection of all the heavy metals used in the antibody panel each time. 
For each IMC ablation, the steps of antigen retrieval and cocktail hybridization will be confirmed by visual inspection of the resident cell type markers using mcd viewer to identify the nuclei, proximal tubules, glomeruli, thick ascending limb, distal convoluted tubules and collecting duct to confirm that the correct region was ablated and analyzed.  

[STEPS]
SECTION: Staining Tissue Sections
1. Bake the slides   at 60°C. Ensure that all visible wax has been removed

SECTION: Staining Tissue Sections
2. Dewax the slides in xylenes in the fume hood for 10 min 

SECTION: Staining Tissue Sections
3. Repeat step 2 and dewax the slides in fresh xylene in the fume hood for 10 min 

SECTION: Staining Tissue Sections
4. Hydrate the slides in descending grades of ethanol (100%, 95%, 80%, 70%) for 5 min each.

SECTION: Staining Tissue Sections
5. Wash the slides in double distilled water for 5 min in a Coplin jar placed on an orbital shaker plate with gentile agitation. 

SECTION: Staining Tissue Sections
6. Perform antigen retrieval bu immersing slides in 1X epitope retrieval bugger (pH 9.0) for 20 min in a steamer

SECTION: Staining Tissue Sections
6.1. Prepare 1X epitope retrieval buffer from its 10X stock solution.
SECTION: Staining Tissue Sections
7. Cool slides for 40 min 

SECTION: Staining Tissue Sections
8. Wash the slides in double distilled water for 5 min in a Coplin jar with gentle agitation on an orbital shaker. Perform this step twice. 

SECTION: Staining Tissue Sections
9. Wash the slides with 1X TBS for 10 min with gentle agitation on an orbital shaker.

SECTION: Staining Tissue Sections
10. Use a PAP pen to draw an outline encircling the sample. 
SECTION: Staining Tissue Sections
11. Block with 3% BSA in 1X TBS for 60 min at room temperature in a hydration chamber.

SECTION: Staining Tissue Sections
11.1. You can use an empty pipette tip box where the slides rest on the tip shelf and the bottom is filled halfway with water. 
SECTION: Staining Tissue Sections
11.2. Blocking solution should be diluted from 10% BSA freshly made from powder. The remaining 10% BSA should be aliquoted and stored at -20°C and diluted at time of use. 
SECTION: Staining Tissue Sections
11.3. Use enough blocking solution to cover the section.
SECTION: Staining Tissue Sections
12. Prepare the antibody cocktail calculating the total volume of antibodies at the concentrations specific for the assay and bring the volume up to a final volume using 0.5% BSA. in 1X TBS.
SECTION: Staining Tissue Sections
12.1. Dilutions can be found on Table 1. 
SECTION: Staining Tissue Sections
13. Place the slides in a hydration chamber and pipette the antibody mix on to the section.
SECTION: Staining Tissue Sections
13.1. Store the antibody cocktail mix on ice and add it on to the samples within 1-2 hours of preparation for best results.
SECTION: Staining Tissue Sections
14. Incubate overnight with the antibody cocktail at 4°C in a hydration chamber.
SECTION: Staining Tissue Sections
15. Wash the slides in 0.1% Triton X-100 in 1X TBS for 5 min in Coplin jars with slow agitation on an orbital shaker. 

SECTION: Staining Tissue Sections
16. Repeat step 15 two more times.
SECTION: Staining Tissue Sections
17. Wash the slides in 1X TBS for 5 min with gentle agitation on an orbital shaker. 

SECTION: Staining Tissue Sections
18. Repeat step 17. 
SECTION: Staining Tissue Sections
19. Stain the tissue with Intercalator-Ir in 1X PBS (1:1000) for 60 min at room temperature in a hydration chamber. 

SECTION: Staining Tissue Sections
20. Wash the slides in double distilled water for 5 min with gentle agitation on an orbital shaker. 

SECTION: Staining Tissue Sections
21. Air dry the slides for at least 20 min at room temperature. 

SECTION: Imaging Mass Cytometry
22. Load the stained slide into the pre-tuned and pre-calibrated Hyperion IMC System.
SECTION: Imaging Mass Cytometry
23. Create a new project on the CyTOF software.
SECTION: Imaging Mass Cytometry
24. Draw a panorama that covers the tissue that will be ablated.
SECTION: Imaging Mass Cytometry
25. Determine regions of interest (ROIs) to ablate with increments less than 3mm2 on the tissue, ensuring that cortical and medullary regions are separate within the panorama created. Regions with artificial section damage are excluded. 
SECTION: Imaging Mass Cytometry
26. Create or select an antibody/metal template. 
SECTION: Imaging Mass Cytometry
27. Make sure to select "generate txt file".
SECTION: Imaging Mass Cytometry
28. Save the project file and start ablation of tissue.
SECTION: Image Verification
29. Once ablation is complete, load the file on MCD viewer to check the quality of each image and channel.