TITLE: Tissue Staining for Imaging Mass Cytometry
AUTHORS: John Herndon, Madelyn Carmody

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the preparation and staining of human FFPE material for multiplex visualization using Imaging Mass Cytometry (IMC). This protocol uses basic standard immunohistochemical staining techniques.  The tissue is incubated with antibodies that have specific affinity for different cells and tissues in the context of a formalin fixed thin section of human tumor material. Antibodies are labeled with heavy metals instead of the typical fluorochromes, and are visualized using the Hyperion Imaging System manufactured by the Fluidigm corporation.</div></div>

[STEPS]
?. Place positively charged slides with    thick tissue section in a   oven for
60 °C
?. Place slides in fresh xylene for
?. RehydratePlace slides in to 100% Ethanol,
?. Antigen RetrievalIncubate slides for   in antigen retrieval solution from step 4Make sure the   is maintained consistently throughout the 30 minute incubation.
96 °C
?. After incubation, cool slides to   on the bench top (about 10 minutes)
70 °C
?. WashPlace slides in  distilled water for   with gentle agitation
?. Place slides in PBS for   with gentle agitation
?. Block Nonspecific StainingEncircle each sample with a PAP pen
?. Blocking solution is 3% BSA in PBS, add just enough to cover the sample and incubate for   at   in a hydration chamber.
0 Room temperature
?. Antibody StainingPipette  pre-prepared antibody cocktail in 0.5% BSA and incubate   at  in hydration chamber
50 µl
4 °C
?. Wash Wash slides with PBS with 0.2% Triton X-100 for   with gentle agitation
?. Wash slides in PBS for   with gentle agitation
?. DNA Intercalator-IrPrepare Iridium intercalator by diluting 1 to 1000 in PBS ( this dilution may have to determined empirically)Pipette just enough iridium intercalator to cover sample and incubate for   at
0 Room temperature
?. Remove iridium intercalator by gently tapping edge of slide to a paper towel
?. Ruthenium Counter StainPrepare a stock solution of Ruthenium Red by mixing 0.0025 grams in PBS, vortex well to mixDilute Ruthenium to working concentration of 0.0005%Counterstain tissue by pipetting just enough of the 0.0005% ruthenium red in PBS for  (duration of ruthenium red may need to be determined empirically)
?. Remove ruthenium red solution by gently tapping edge of slide to paper towel
?. Wash slides with PBS for  with gentle agitation
?. Wash Wash slides with distilled water for   with gentle agitation
?. Air dry slides at room temperature for
?. Repeat Step 20,
?. repeat step 22,
?. repeat Step 13,
?. repeat Step 15
?. Slides are now ready for Hyperion Imaging.
?. Places slides in to 95% Ethanol,
?. Placed slides in to 80% Ethanol,
?. Place slides in to 70% Ethanol,
?. Turn on Water bath for Antigen Retrieval,
96 °C
?. Place slides in to distilled water for * Note slides may be kept for a longer period at this step, make sure the water bath is the correct temperature before proceeding to the next step.
?. Prepare Antigen retrieval solution of Tris EDTA  with 10% Glycerol
?. Place container for slides with above antigen retrieval solution in to water bath to pre-warm