TITLE: FFPE Tissue Pre-treatment Before t-CyCIF on Leica Bond RX
AUTHORS: Jia Ren Lin, Benjamin Izar, Zoltan Maliga, Yu-An Chen, Giorgio Gaglia, Ziming Du, Clarence Yapp, Shaolin Mei, Sandro Santagata, Peter Sorger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tissue-based cyclic immunofluorescence (t-CyCIF) is optimized for FFPE specimens mounted on glass slides. Dewaxing and antigen retrieval are important steps to remove wax and expose antigenic sites. This protocol describes dewaxing and antigen retrieval on a Leica Bond RX automated slide processor; similar instruments are manufactured by Ventana or Dako and are commonly found in histopathology core facilities. t-CyCIF can also be performed following manual de-waxing and antigen retrieval (e.g. microwaving slides in citrate buffer or using a pressure cooker).</div></div>

[STEPS]
?. Create a protocol file named "t-CyCIF" on the Leica Bond RX (see user manual for instrument-specific details):
?. Bake FFPE slides at  for .
60 °C
?. Dewax by rinsing three times with  preheated () Bond dewax solution, following by 3 rinses of , 200 proof ethanol.
150 µl
60 °C
150 µl
?. Remove the Bond dewax solution. Add  Bond ER1 solution for antigen retrieval and incubate at  for .
150 µl
99 °C
?. Remove the Bond ER1 solution and block for  with  Intercept® (PBS) Blocking Buffer at .
150 µl
0 Room temperature
?. Remove the blocking buffer and incubate with  secondary antibody solution by incubating for  at .
150 µl
0 Room temperature
?. Remove the secondary antibody solution and incubate with  Hoechst solution for  at .
150 µl
0 Room temperature
?. Prepare reagent chambers for Leica Bond RX. CRITICAL STEP Prior to the first cycle of t-CyCIF, a prestaining (blocking) step is performed by incubating tissues with a mixture of appropriate secondary antibodies to block non-specific binding sites in the tissue. Secondary antibodies are chosen based on the species and isotypes of the unconjugated antibodies used in the first t-CyCIF cycle. CRITICAL STEP Avoid using Alexa Fluor 546-, Alexa Fluor 568- or Alexa Fluor 594-conjugated secondary antibodies, as these fluorophores are resistant to bleaching.
?. Fill chamber 1 with  of 1X PBS.
30 mL
?. Fill chamber 2 with  of Intercept® (PBS) Blocking Buffer.
7 mL
?. Fill chamber 3 with of the appropriate secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 diluted in Intercept® (PBS) Blocking Buffer (1:1000, vol:vol).
2 mL
?. Open the lids of the chambers and put them in the chamber tray in the Leica Bond RX.
?. Place chambers in reagent tray and insert into Leica Bond RX.
?. Create a new study on the Leica Bond RX, select “t-CyCIF” file created as the protocol, add each slide to the study, and print labels for each FFPE slide. The Bond requires barcoded labels on all slides to process them.
?. Place all labeled slides onto a slide tray, cover the slides with Bond Universal Covertiles, and insert the slide tray into the machine.
?. The machine will scan the barcodes on the stickers of the slides and reagent chambers. Check the label readings by hovering the mouse over the slide labels. If all of the slides have been recognized correctly, then click the START button to run the protocol. This will take approximately .
?. Remove slides from the Leica Bond RX and place them into 1X PBS.■ PAUSE POINT Slides can be stored in 1X PBS at  for several days after processing on the Bond RX. Ensure that the entire tissue is covered in 1X PBS; otherwise the tissue will dry out and yield poor results.
4 °C