TITLE: Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval
AUTHORS: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to manually deparaffinize and rehydrate slides for the Cell DIVE™ Platform.</div></div>

[STEPS]
?. [Slide Baking]
This is the initial step of the deparaffinization process. This step serves two purposes: (1) melting of the paraffin wax and (2) ensuring tissue adherence to the slides.
?. [Slide Baking]
Set the incubator to .
60 °C
?. [Slide Baking]
Load tissue slides into a slide rack. All tissue slides should have the tissue side facing up and parallel to the rack.
For fragile/fatty tissues, the slides may be placed in front of a fan overnight, before slide baking, to remove excess moisture.
?. [Slide Baking]
Place slide rack (with tissue side facing up) into a incubator.
60 °C
?. [Slide Baking]
Incubate for at least . We recommend  baking for optimal tissue retention.
?. [Reagent Preparation]
After placing FFPE slides in the incubator for baking, prepare the following reagents – xylene, 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol, 1X PBS, and 1X PBS with 0.3% Triton X100.
Reagent amounts ( ) are for a staining dish that can contain up to 24 slides.
If only 5 slides, use a coplin jar and make up all of the working solutions to a   final volume.
Make sure to fill up the container only up to frost edge (lower edge). Avoid pouring solutions all the way up to the top of the slide (labels and pen writing on frosted edge may contaminate samples).
?. [Reagent Preparation]
Before preparing reagents, place blue underpads on the bench to absorb spills.
?. [Reagent Preparation]
After baking, follow steps below.
?. [Reagent Preparation]
Transfer slides to xylene and wash,  each, with gentle agitation (1/2).
?. [Reagent Preparation]
Place slides in 100% ethanol and wash,  each with gentle agitation (1/2).
?. [Reagent Preparation]
Place slides in 95% ethanol and wash,  each with gentle agitation (1/2).
?. [Reagent Preparation]
Place slides in 70% ethanol and wash,  each with gentle agitation (1/2).
?. [Reagent Preparation]
Place slides in 50% ethanol and wash,  each with gentle agitation (1/2).
?. [Reagent Preparation]
Place slides in PBS and wash for  each with gentle agitation (1/2).
?. [Reagent Preparation]
Permeabilize tissue in 1X PBS with 0.3% Triton X100 for .
?. [Reagent Preparation]
Wash in 1X PBS for .
During this wash, begin pre-warming the antigen retrieval solutions that are required for the next step. Refer to the "Antigen Retrieval" Section for additional information regarding solution prep.
?. [Antigen Retrieval]
During slide clearing and hydration prepare the reagents needed for antigen retrieval (ARS1+ARS2):
?. [Antigen Retrieval]
Prepare ARS1 (Citrate based solution):
?. [Antigen Retrieval]
• Label a beaker or 250ml cylinder “ARS1”
?. [Antigen Retrieval]
Label a beaker or 250ml cylinder “ARS1”.
?. [Antigen Retrieval]
Add  to the beaker.
[ddH20]
?. [Antigen Retrieval]
Remove Vector Labs citrate antigen retrieval solution from 4°C storage.
?. [Antigen Retrieval]
Shake citrate solution vigorously.
?. [Antigen Retrieval]
Add  to ARS1 beaker.
[citrate solution]
?. [Antigen Retrieval]
Add a magnetic stir bar, place on magnetic stirrer, and stir thoroughly (if using a graduated cylinder, cover with parafilm and invert to mix).
?. [Antigen Retrieval]
Transfer to a metal staining dish from the NxGen Decloaker labeled “ARS1.”
?. [Antigen Retrieval]
pH using Biocare pH strips, verify pH is .
?. [Antigen Retrieval]
Prepare ARS2 (Tris based solution).
?. [Antigen Retrieval]
Label a beaker or 250ml cylinder “ARS2”.
?. [Antigen Retrieval]
Add  to the beaker.
[ddH20]
?. [Antigen Retrieval]
Once the aliquot of 10x ARS2 has thawed, add it to the beaker. If the aliquot is partially thawed, you can pour the frozen solution into the water.
?. [Antigen Retrieval]
Add a magnetic stir bar, place on magnetic stirrer, and stir thoroughly. If using a graduated cylinder, cover with parafilm and invert to mix.
?. [Antigen Retrieval]
Transfer to a metal staining dish from the NxGen Decloaker labeled “ARS2.”
?. [Antigen Retrieval]
pH using Biocare pH strips, verify pH is between  and .
?. [Antigen Retrieval]
Prepare the Biocare NxGen Decloaker.
?. [Antigen Retrieval]
Add  to the bottom of pressure cooker.
[DI water]
?. [Antigen Retrieval]
Be sure to add fresh DI water before each antigen retrieval run.
?. [Antigen Retrieval]
Fill 3 metal staining dishes (provided with the Biocare NxGen decloaker) containing the two antigen retrieval solutions (ARS1 and ARS2) and DI water ().
250 mL
The maximum amount of slides per run is 24.
pH - Citrate should be 6 and TRIS should be 8.5/9.
?. [Antigen Retrieval]
Place the staining dishes containing the antigen retrieval solutions in the wire rack in the outside positions and the staining dish containing the water in the center position.
?. [Antigen Retrieval]
Place the wire rack with the staining dishes into the water bath.
?. [Antigen Retrieval]
Plug the decloaker into a wall outlet to turn the unit on – it is not equipped with a power switch. The display screen will turn on.
?. [Antigen Retrieval]
Press the lower left hand button (Menu button: page with lines) once and a list of temperatures will appear. Select  using the menu button (lower left) and press the green circle (center button) once.
110 °C
?. [Antigen Retrieval]
Set the time to  using the lower menu and timer buttons as indicated on the screen and press the green button to accept.
?. [Antigen Retrieval]
The pressure cooker is now waiting to start and can sit in this state until the slides are ready.
?. [Antigen Retrieval]
Complete the below checklist and include it in your documentation for the Antigen Retrieval run.  ABC1Pre-Warming Antigen Retrieval ChecklistDuration (min)Comments2Added 500mL of fresh DI water to the
pressure cooker?5 minutes3ARS1 and ARS2 prepared appropriately?Consult working solutions section.4pH’d both ARS1 and ARS2 prior to heating?ARS1 pH should be 6, ARS2 pH should be 8.5/9.
ABC1Pre-Warming Antigen Retrieval ChecklistDuration (min)Comments2Added 500mL of fresh DI water to the
pressure cooker?5 minutes3ARS1 and ARS2 prepared appropriately?Consult working solutions section.4pH’d both ARS1 and ARS2 prior to heating?ARS1 pH should be 6, ARS2 pH should be 8.5/9.
?. [Antigen Retrieval]
Place the lid on the Decloaking chamber and make sure it is in the locked position (metal button over the left handle); turn lid towards you to lock. Position the pressure limit valve as directed in image below, ensuring it is set level on the pressure stem, not tipped to one  side.
?. [Antigen Retrieval]
Press the center green button once to begin the program. When the temperature display on the front of the decloaker shows  start an external timer for .
70 °C
The slides will spend 20 minutes in ARS1, 20 minutes in ARS2, and then sit for 10 minutes on the benchtop in ARS2. The program will count down 4 minutes once the pressure cooker has reached the set temperature of 110°C. The external timer is used to time the 20 minutes required in ARS1 since the program does not do this.
?. [Antigen Retrieval]
Pressure cooker will hold at 110 °C for . Once the 4 min hold is complete, record the temperature and pressure from the device display on the log sheet. Leave the slides in ARS1 solution for a total time of  (until the external timer beeps). Over the remaining time, the temperature should decrease.
?. [Antigen Retrieval]
After the external timer has beeped, record the displayed temperature and pressure on the log sheet.
?. [Antigen Retrieval]
Carefully open the lid (use gloves as the surface is hot). Water will run from the lid when tipped so be sure to not allow the water to pour into either of the AR solutions. Remove steam strip indicator, steam strip should be uniformly brown if passed (light brown is OK). Allow steam strip to dry and then tape into log sheet.
?. [Antigen Retrieval]
Transfer the slides into Tris-Antigen Retrieval working solution (ARS2) and close pressure cooker (it will not lock after the run is complete, but it will slowly cool down even though it is not in the locked position).
?. [Antigen Retrieval]
Restart the external timer for . During the 20 minutes, the decloaker will beep to indicate the run has been completed. Press the green circle (center button) to stop the beeping, but leave the decloaker closed and slides in ARS2. The temperature will decrease during this time.
?. [Antigen Retrieval]
After the external timer has beeped, open pressure cooker, and record the final displayed temperature and pressure on the log sheet.
?. [Antigen Retrieval]
Check pH of solutions with test strips and record on log sheet. For the pH, AR solution 1 (citrate) should be  and AR solution 2 (Tris) should be / .
?. [Antigen Retrieval]
Remove the wire rack and place on the bench for  (keep slides in hot Tris-ARS2 during this cool-down).
?. [Antigen Retrieval]
Let the pressure cooker cool down to . Unplug the pressure cooker to turn it off.
50 °C
?. [Antigen Retrieval]
Wash slides ( each) in PBS with gentle agitation on shaker (1/4).
?. [Antigen Retrieval]
Proceed to slide blocking.
?. [Slide Blocking]
During slide clearing and hydration prepare the reagents needed for slide blocking.
?. [Slide Blocking]
Remove two  from the -20°C freezer.
[aliquots of reconstituted donkey serum]
?. [Slide Blocking]
Allow the donkey serum to thaw at .
50 Room temperature
?. [Slide Blocking]
Prepare Blocking Solution. Let the slides sit in 1X PBS until the blocking solution is ready.
?. [Slide Blocking]
Label a beaker “Blocking Solution.”
?. [Slide Blocking]
Add  to the beaker.
[1x PBS]
?. [Slide Blocking]
Weigh out and add  to the beaker.
[Bovine Serum Albumin (BSA)]
?. [Slide Blocking]
Add a magnetic stir bar, place on magnetic stirrer, and stir thoroughly.
?. [Slide Blocking]
While stirring, add the 2x  to the beaker.
[aliquots of donkey serum]
?. [Slide Blocking]
Continue to mix thoroughly.
?. [Slide Blocking]
Cover with parafilm and leave at  until you are ready for slide blocking.
50 Room temperature
?. [Slide Blocking]
Once you’re ready for slide blocking, pour the prepared Slide Blocking Solution into a staining dish and transfer slides from 1X PBS into the Slide Blocking staining dish.
?. [Slide Blocking]
Incubate for at least  at , or  at .
50 Room temperature
4 °C
?. [Slide Blocking]
After the incubation, wash slides one time with fresh 1X PBS for  on an orbital shaker set to 60 RPM.
?. [Initial DAPI Staining]
If DAPI working solution is not available, prepare it as per Table 5 in the "Reagent Prep" Section.
?. [Initial DAPI Staining]
Pour DAPI solution into a staining dish.
?. [Initial DAPI Staining]
Transfer blue slide rack (containing the slides) sitting in 1X PBS into the “DAPI” staining dish.
FFPEs are stained with DAPI to label the cell nuclei for imaging purposes.
?. [Initial DAPI Staining]
Incubate slides in DAPI working solution for  at  with gentle agitation on an orbital shaker set to 60 RPM.
50 Room temperature
?. [Initial DAPI Staining]
After the 15 minute incubation, transfer the slide rack into a staining dish containing fresh 1X PBS.
Do not discard the DAPI solution. The DAPI may be reused up to 10 times or by the expiration date, whichever comes first. Tally the number of uses on the DAPI bottle.
?. [Initial DAPI Staining]
Wash slides 1x for  in fresh 1X PBS with gentle agitation on an orbital shaker set to 60 RPM.
?. [Initial DAPI Staining]
Transfer slides to staining dish with dH20 and proceed to coverslipping.
?. [Coverslipping Slides]
Mounting media should be removed from the 4°C fridge and brought to .
50 Room temperature
?. [Coverslipping Slides]
Take a rainin pipet tip (200uL-green box) and with a scissors clip the end to make a wide bore.
?. [Coverslipping Slides]
Take up  (4% propyl gallate, 1% DABCO, 90% glycerol) and add to one end of the slide. For leeching markers, use alternate mounting media (50% glycerol, 4% propyl gallate).
[mounting media]
?. [Coverslipping Slides]
Place the end of the coverslip at a 45 degree angle and slowly lower; allowing the mounting media to flow under the coverslip across the slide.
?. [Coverslipping Slides]
Check to make sure there are no bubbles over the tissue. Also make sure there is not mounting media oozing out of the sides of the coverslip; remove any excess.
?. [Coverslipping Slides]
Proceed to Background Imaging or store the slides at  in light protected environment.
4 °C
?. [Antigen Retrieval]
Remove a  aliquot of the 10x Tris Antigen Retrieval Solution (ARS2) from the -20°C freezer.
25 mL
?. [Antigen Retrieval]
Allow the ARS2 to thaw at .
-20 Room temperature
?. [Antigen Retrieval]
?. [Reagent Preparation]
Transfer slides to xylene and wash,  each, with gentle agitation (2/2).
?. [Reagent Preparation]
Place slides in 100% ethanol and wash,  each with gentle agitation (2/2).
?. [Reagent Preparation]
Place slides in 95% ethanol and wash,  each with gentle agitation (2/2).
?. [Reagent Preparation]
Place slides in 70% ethanol and wash,  each with gentle agitation (2/2).
?. [Reagent Preparation]
Place slides in 50% ethanol and wash,  each with gentle agitation (2/2).
?. [Reagent Preparation]
Place slides in PBS and wash for  each with gentle agitation (2/2).
?. [Antigen Retrieval]
Wash slides ( each) in PBS with gentle agitation on shaker (2/4).
?. [Antigen Retrieval]
Wash slides ( each) in PBS with gentle agitation on shaker (3/4).
?. [Antigen Retrieval]
Wash slides ( each) in PBS with gentle agitation on shaker (4/4).
?. [Antigen Retrieval]
Place slides in the Citrate-Antigen Retrieval working solution (ARS1). Place steam test strip on the center staining (water) dish so that it rests across the top of the staining dish without touching liquid. Please note that the metal staining dishes do not have lids.