TITLE: Fresh frozen tissue staining with CODEX tagged antibody panel
AUTHORS: Yury Goltsev, Nikolay Samusik, Julia Kennedy-Darling, Salil Bhate, Matthew Hale, Gustavo Vazquez, Sarah Black, Garry Nolan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>CODEX is a technology that uses oligo labeled antibodies, specialized fluorescent probes, and a companion instrument along-side a standard fluorescence microscope to create single-cell resolution fluorescence data across a multitude of parameters within spatial context in a single tissue.  CODEX was developed by Yury Goltsev and Nikolay Samusik in the laboratory of Garry Nolan at Stanford. The technology is being commercialized by </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Akoya Biosciences</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Tissue Preparation]
Begin with fresh frozen tissues sectioned onto 22mm2  poly-lysine coated coverslips and stored at -80C.  Add a 5mmlayer of drierite granules on the bottom of a pipet tip box or similar container.  Place a piece of kimwipe on top of the drierite.  Retrieve the coverslips from freezer with bent-tip tweezers and place on top of the kimwipe, tissue-side-up.A scratch with the tip of the tweezers on the OCT layer can determine which side of the coverslip has the tissue.Close the box and let sit at RT for 2 minutes.
?. [Tissue Preparation]
Transfer coverslips to a coverslip staining jar containing acetone.  Incubate at RT for 10 minutes.
?. [Tissue Preparation]
Arrange coverslips on a paper towel, tissue-side-up, and allow to air-dry for 2 minutes.
?. [Tissue Preparation]
Fill the wells of a 6-well TC plate with ~5ml Staining Buffer 1 and transfer the coverslips into the wells for the first rehydration step. Incubate for 2 minutes.
?. [Tissue Preparation]
Fill the wells of another 6-well TC plate with ~5ml Staining Buffer 1 and transfer the coverslips into the wells for a second rehydration step. Incubate for 2 minutes.The first 6-well TC plate can be rinsed out with ddH2O and re-used.  Several TC plates can be rinsed, re-used, and rotated for the duration of the protocol.
?. [Tissue Preparation]
Prepare fixing solution in a conical tube by making a 10-fold dilution of 1.6% PFA in Staining Buffer 1.  Each coverslip will require 2ml of fixing solution.
?. [Tissue Preparation]
Add 2ml fixing solution to new wells and transfer the coverslips into the fixing solution.  Incubate for 10 minutes.
?. [Tissue Preparation]
After the fixation, transfer the coverslips to wells containing ~5ml Staining Buffer 1.
?. [Tissue Preparation]
Transfer the coverslips to new wells containing ~5ml Staining Buffer 1.
?. [Tissue Preparation]
Transfer the coverslips to new wells containing ~5ml Staining Buffer 2, and allow to equilibrate for at least 2 minutes.  Tissues can stay in this solution for up to 30 minutes while antibody cocktail is being prepared.
?. [Antibody Cocktail Preparation]
The antibody cocktail is a combination of all the antibodies in the panel plus CODEX Blocking Buffer.  Prepare 200ul CODEX Blocking Buffer for each coverslip to be stained.180ul Staining Buffer 25ul Blocking component 15ul Blocking component 25ul Blocking component 35ul Blocking component 4200ul total for each coverslip
?. [Antibody Cocktail Preparation]
The antibody volume needed per coverslip should have been determined by prior titration experiments.  A good starting volume is 1ul per coverslip in a 200ul cocktail.Determine the total antibody volume that will go into the cocktail.  The antibody cocktail is the entire antibody panel volume plus a volume of CODEX Blocking Buffer  to equal 200ul.   example:        27ul antibodies + 173ul CODEX Blocking Buffer  = 200ul antibody cocktail                        18ul antibodies + 182ul CODEX Blocking Buffer  = 200ul antibody cocktail
?. [Antibody Cocktail Preparation]
Combine antibodies and CODEX Blocking Buffer  and gently mix with pipet.
?. [Staining]
Remove coverslips from Staining Buffer 2, dab the corner on a kimwipe to remove most of the liquid, place tissue-side-up on top of the humidity chamber, and pipet 190ul antibody cocktail on top.  Take care to not pipet directly on top of the tissue.  Surface tension should hold the entire volume on top of the coverslip.  Do not allow tissues to dry out.
?. [Staining]
Close the humidity chamber and incubate at RT for 3 hours.
?. [Post-processing of Stained Tissue]
Take coverslips, dab a corner on a kimwipe to remove most of the liquid, and place into wells containing ~5ml Staining Solution 2.  Incubate 2 minutes.
?. [Post-processing of Stained Tissue]
Transfer coverslips to new wells with Staining Solution 2 for a total of 2 washes.Incubate 2 minutes.
?. [Post-processing of Stained Tissue]
Prepare fixing solution in a conical tube by making a 10-fold dilution of 1.6% PFA in Staining Buffer 3.  Each coverslip will require 2ml of fixing solution.
?. [Post-processing of Stained Tissue]
Add 2ml fixing solution to new wells and transfer the coverslips into the fixing solution.  Incubate for 10 minutes.
?. [Post-processing of Stained Tissue]
During the fixation, place a 6-well TC plate on a bed of slushy ice.  Allow the plate to chill for 2 minutes and then add ~5ml of ice cold (4C) methanol to one well per coverslip.
?. [Post-processing of Stained Tissue]
After the 10 minute fixation, transfer the coverslips to wells containing ~5ml PBS.Transfer the coverslips to new wells containing PBSTransfer the coverslips to new wells containing PBS, for a total of 3 washes.
?. [Post-processing of Stained Tissue]
Transfer the coverslips to the ice cold methanol.Incubate for 5 minutes.
?. [Post-processing of Stained Tissue]
After the 5 minute incubation, transfer the coverslips to wells containing ~5ml PBS.  Methanol dries fast so this step should be done quickly.  Ensure that the coverslip sinks all the way to the bottom of the well.Transfer the coverslips to new wells containing PBSTransfer the coverslips to new wells containing PBS, for a total of 3 washes.
?. [Post-processing of Stained Tissue]
Prepare 200ul Final Fixative Solution for each coverslip.  One aliquot of CODEX Final Fixative (F) yields 1ml Final Fixative Solution for 5 coverslips.Cut aliquots from the Final Fixative (F) strip, allow to thaw, and quickly spin down.
?. [Post-processing of Stained Tissue]
Pipet 20ul Final Fixative (F) into 1ml PBS.  Gently mix with pipet.  ABC1Final Fixative Solution1-5 coverslips6-10 coverslips2PBS1000ul2000ul3(F) Aliquots20ul40ul
ABC1Final Fixative Solution1-5 coverslips6-10 coverslips2PBS1000ul2000ul3(F) Aliquots20ul40ul
?. [Post-processing of Stained Tissue]
Rinse and dry humidity chamber tray to remove any residual antibodies from the staining.  Take the coverslips, dab the corners on a kimwipe, and place them on the humidity chamber.
?. [Post-processing of Stained Tissue]
Add 190ul Final Fixative Solution on top of each coverslip, taking care to not pipet directly on the tissue.  Ensure that the entire tissue is covered in solution.  Close humidity chamber.Incubate for 20 minutes.
?. [Post-processing of Stained Tissue]
After the 20 minute fixation, transfer the coverslips to wells containing ~5ml PBS.Transfer the coverslips to new wells containing PBSTransfer the coverslips to new wells containing PBS, for a total of 3 washes.
?. [Post-processing of Stained Tissue]
Store the stained and fixed samples in a 6-well TC plate in ~5ml Staining Buffer 3 at 4C for up to 2 weeks.