TITLE: Imaging Mass Cytometry Antibody Staining 
AUTHORS: Michelle Daniel, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following SOP describes procedures required for the antibody staining of formalin fixed,</div><div class = "text-block">paraffin-embedded (FFPE) samples. In this SOP, the term “samples” refers to FFPE</div><div class = "text-block">tissue samples.</div></div>

[STEPS]
?. [Sample de-paraffinization and rehydration of samples]
1. Unstained samples should have time to equilibrate to room temperature for at least 10 min beforeproceeding to the next step.2. Take the unstained samples to the dewaxing robot3. Log use of dewaxing robot in log sheet4. Make sure the levels of all reagents in the containers are sufficient. Refresh reagents if longerthan 5 days in use and/or if used more than 5 times.5. Press the button to lift the sample holder arm and load the unstained samples into the slideholderand place the slideholder in the sample holder.6. Choose program 1.7. Start the program.8. After the program finished, press button to lift arm, transfer slideholder with the slides into a traycontaining TBS buffer.
Perform these steps in fume hood.
?. [Epitope retrieval (HIER) and slide staining with antibody mixture]
1. Prepare decloaking chamber. Add 500 mL of ddH2O to the decloaking chamber, fill all cuvettesthat are not used with 200 mL ddH2O and those that will be used with 200 mL of HIER buffer.2. Select the protocol (30 min, 95 °C) and start the program to heat up the chamber.3. Once the temperature reaches 95 °C a message will appear on the display prompting the user toload the slides. Open the pressure chamber carefully and place the slideholder with the slidesfrom step 3.3.8 in the tray with HIER buffer.4. Incubate at 95 °C for exactly 30 min.5. Open the pressure chamber carefully, place the tray containing the slideholder on bench at roomtemperature and cool down for 20 min.6. Transfer the slides into TBS buffer.
?. [3.4 Blocking of unstained samples]
1. Remove one slide from TBS, tap sideways/gently flick to remove excess buffer, circle eachsample on the slide with a hydrophobic barrier pen and place the slide in a wet-chamber.Caution! If there are multiple samples on the slide make sure that none of them dries out duringhydrophobic barrier creation. You may add some TBS on top of the samples to prevent this.2. Repeat step 1 for all slides.3. When all tissues have been circled with a pap pen tap sideways or gently flick the slide to removethe TBS and add 100 μL of blocking buffer to each sample. Process one slide after the other.4. Incubate for 1h at room temperature in wet-chamber.
It is crucial that during the next procedure the biological material on the slide never dries out.
?. [Antibody Staining]
1. Prepare the antibody mix. Fill up to the final volume with antibody diluent.        a. If you have a production panel or many antibodies, use the 2x aliquots to prevent a dilutionwith antibody medium from the antibodies used. Dilute your mix with TBS accordingly sothat your mix has the correct final concentration.2. Add a sufficient volume of the antibody mix to cover the tissue (50 μL/Sample).3. Close the wet-chamber and move to 4 °C. Ensure tissue is not drying out during incubation bychecking that the incubation chamber is completely leveled. Incubate overnight.
?. [DNA Staining and Sample Drying ]
1. Retrieve wet-chamber from 4 °C.2. Tap sideways/gently flick slides and place in TBS using the tray and the slideholder specific forthe slide wash robot.3. Fill cuvettes from wash station with TBS and start program TBSwash4. During washing, prepare Ir-intercalator solution (50 μL/Sample).5. Incubate slides with Ir-intercalator solution for 5 min in wet-chamber.6. Wash slides 3x5 min in TBS as in step 3.7. Dip slides with stained samples briefly in ddH2O.8. Dry slides with stained samples under airflow.9. Store slides with stained samples in a box at room temperature.