TITLE: CODEX Acquisition Protocol
AUTHORS: Franchesca Farris, Marda Jorgensen, Jerelyn Nick, Jesus Peñaloza

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"> Multiplex imaging of lymph node, thymus and spleen is accomplished using the Akoya Biosciences CODEX system at the  HubMAP Tissue Mapping Center at the University of Florida, TMC-UF.</div><div class = "text-block">This protocol is an overview of the CODEX raw data acquisition process guided by the CODEX Instrument Manager (CIM) , which is part of the CODEX Software Suite. The CODEX Instrument Manager is necessary to perform CODEX experiments. It controls the fluidics of the CODEX instrument the integration and synchronization with the Keyence microscope, image data formatting and facilitates transfer of data to the analysis computer.</div><div class = "text-block">For more information, consult the Akoya Biosciences CODEX User Manual - A.0.</div></div>

[STEPS]
?. [loading a blank coverslip and priming the instrument]
:Untwist the two wing knobs on the coverslip stage to remove the lid holding the fluidic line
?. [loading a blank coverslip and priming the instrument]
If running 2 experiments back to back, first remove old cover slip, using bent angle forceps. Rinse area well with deionized water. Wipe dry with kimwipe.
?. [loading a blank coverslip and priming the instrument]
Soak two gaskets in 1X CODEX buffer for at least 10 minutes.
?. [loading a blank coverslip and priming the instrument]
Place the first gasket into the divots at the center of the stage. Tap gently with forceps to be sure it is well seated.  Place a clean coverslip (required for priming)  on top of the first gasket. If a sample coverslip is inserted, confirm that the tissue is facing upwards. Tap the edges of the coverslip to make sure it is adhered to the first gasket. Quickly place the second gasket on top of the coverslip.
?. [Set up CODEX experiment]
Quick Start Experiment Setup:  Turn on CIM computer and CODEX robot
?. [loading a blank coverslip and priming the instrument]
Check that the gaskets and coverslip are inserted squarely into the stage. Place the metal top plate on the stage insert and lock it in place by turning the wing knobs.
?. [loading a blank coverslip and priming the instrument]
Place loaded stage insert into dedicated holder to support it and keep it level during priming.
?. [Set up CODEX experiment]
Launch Codex Instrument Management Software.
?. [Set up CODEX experiment]
Select the Experiment Tab to start the run.
?. [Set up CODEX experiment]
Setup the CODEX instrument  with the start-up Wizard
?. [Set up CODEX experiment]
Select New Template for inputting experimental settings from scratch or Open Template to modify an exiting template,
?. [Set up CODEX experiment]
Input the Project and Experiment name. Use the naming format of: src_CX____
?. [Set up CODEX experiment]
Input the Start Cycle Well and Number of Cycles in agreement with the 96-well reporter plate to be used for this experiment.
?. [Set up CODEX experiment]
Within the table, for each cycle, enter the Marker Name, Exposure time, and Class. Marker names should follow the convention agreed to by the HubMap Consortium
The first and last cycles contain nuclear stain only and are labled BLANK for both marker and class. Any unused wells (no marker) are labeled EMPTY for both marker and class.
?. [Set up CODEX experiment]
Select the number of Z-Planes to image. For a 5um section, 11 planes are standard. An uneven section, large region of acquisition or thicker section all require increases in number of Z-planes.The software will alter this number based on the formula provided below the input box, resulting in a number that is less than the set.point
?. [Set up CODEX experiment]
Enter the Operator Name.
?. [Set up CODEX experiment]
Click Validate Experiment to confirm there are no errors within the experimental design.
?. [Set up CODEX experiment]
Press Save Template or Save Template As to save the experimental setting if needed, Save Template As can be used to avoid over writing previous settings.
?. [Set up CODEX experiment]
Press Start Experiment.
?. [Set up CODEX experiment]
The initial pop-up will describe each of the steps used in the Wizard. Press Next.
?. [loading a blank coverslip and priming the instrument]
Prepare Stage Insert for priming the instrument. Follow the steps below to load a blank coverslip.
?. [loading a blank coverslip and priming the instrument]
After setting up the stage with a blank coverslip, Click next to initiate the start-up wizard.
?. [loading a blank coverslip and priming the instrument]
Select next. The wizard will prompt you to Prime Instrument, click Prime Instrument to initiate procedure.
?. [Load and DAPI stain sample coverslip]
The wizard will prompt performing Nuclear Staining of tissue on coverslip.
?. [Load and DAPI stain sample coverslip]
Remove DAPI Nuclear Staining stock solution from 4C storage and place it on ice.
?. [Load and DAPI stain sample coverslip]
Label a 1.5ml amber microfuge tube for Nuclear Stain Solution. Add 1 mL of 1x CODEX Buffer and 0.5ul of DAPI, invert to mix.
?. [Load and DAPI stain sample coverslip]
Remove the blank coverslip and load a sample coverslip in to the stage insert as in step 3.4..  Quickly add 700uL of the Nuclear Stain Solution to the sample well and protect it from light.
Be sure that the sample coverslip is face up in the stage insert.  Avoid pipetting directly on the tissue. Do not allow the tissue to dry out. Use a moist kim wipe to clean the underside of the sample coverslip after completing the nuclear stain, but before placing the stage insert into the microscope.Dry thoroughly.
?. [Load and DAPI stain sample coverslip]
Press Wait & Wash Tissue. The instrument will perform a 5 minute incubation before washing the sample well.
If the 5 minutes incubation time has passd, click Immediately Wash Tissue to remove the Nuclear Stain solution.
?. [Post Run Clean Instrument Wash]
Post run wash.
This should be performed immediately following every CODEX run.  It will take approximately 5 minutes  to complete.
?. [Post Run Clean Instrument Wash]
Remove the stage insert from the microscope and place it in the dedicated holder. Make sure that the Water Bottle has at least of 20mL of distilled water. Run Clean Instrument Wash from the Maintenance Tab. When the wash run is completed, remove the 96-well plate, empty and clean the reservoirs and all bottles. Close the Driver software and turn off the CODEX instrument.
DMSO is present in all reservoirs and in the waste bottle, dispose of liquids properly.
?. [Maintenance Wash]
Maintenance Wash
?. [Maintenance Wash]
Maintenance Wash should be performed Weekly during daily use of the instrument or Monthly for less frequent use of the instrument. A blank coverslip needs to be in the stage insert. Both Bottle 1 and Bottle 2 caps are placed in the water bottle. This will ensure full cleaning of all lines.
?. [Maintenance Wash]
Select Maintenance Wash from  the Maintenance tab.
?. [Maintenance Wash]
When the Maintenance wash is completed return the fluid lines to their respective bottles.  Remove and discard  the coverslip. Wash the gaskets the stage insert with distilled water. Store them dry and covered with a kim wipe to avoid dust.
?. [loading a blank coverslip and priming the instrument]
Click Check Fluidics.
?. [Maintenance Wash]
Empty waste bottle. Turn off robot.Close CODEX driver software and power off the CIM computer.
?. [loading a blank coverslip and priming the instrument]
Fill the water bottle with distilled  water, at least 100mL. Fill bottle 2 with DMSO and fill Bottle 1 with 1x CODEX buffer according to the set-up chart in Guidelines &Warnings. Make sure to reconnect all lines and to firm all caps on the corresponding bottles.
?. [Microscope set up start acquisition]
Set the  z-plane minimum and maximum values. The exact planes used for each tile will be determined through auto focus by the CIM driver software during image acquisition..
Confirm the pitch is set to 1.5 and define a stitch area ( i.e. 9X9). Set the Z-planes  so that the best focus is the middle point  of the Z-stack.  Check multiple areas of the image area to be sure that the Z-stack has sufficient depth to capture  the best focus over the entire image.
?. [Microscope set up start acquisition]
Create the folder for collecting and storing the raw images.  Available folders are  preloaded in the lab share drive "CODEX Runs" folder.Identify the specific data set using the experimental name designated in step 1.4.
?. [Microscope set up start acquisition]
The wizard will prompt the microscope set up.  Turn on the power supply to the BZ-X800 Keyence microscope. Turn on the microscope.
?. [Microscope set up start acquisition]
Open the microscope viewer software and login with lab designated PassWord.
?. [Microscope set up start acquisition]
Confirm that the saved settings are compatible with the Codex software: Stage holder= versatileMultichannel on, all four channels set to white color and activeHigh resolution,  low photobleaching, Monocromatic, Stitching, Z-stacking all "ON"Set capture area to 7X9, pitch to 1.5, number of Z-planes to match number calculated in step 1.7.
?. [Microscope set up start acquisition]
Remove the 10x and 40x objectives from the microscope.  Set the microscope to have the 20x objective in position for use. Place the liquid overflow detector in the objective turret with the cord trailing from the left side stage cut out.
?. [Microscope set up start acquisition]
Confirm placement of fluorescent filter cubes: DAPI in position 1, AF750 in position 2,  ATTO550 in position 3, and Cy5 in position 4.
?. [loading a blank coverslip and priming the instrument]
Check that  the waste bottle is empty and the 4 buffer reservoirs located inside the instrument are empty and clean.
?. [Microscope set up start acquisition]
Place the Stage Insert into the microscope stage holder.  Activate the DAPI channel and auto focus on the center of the tissue sample.
?. [Microscope set up start acquisition]
Use the Navigation function of the microscope to select imaging region(s). Capture an image of the stitch area and save it to the folder created for this data set. Confirm that the image routed to the correct folder.
?. [Microscope set up start acquisition]
Switch back to the CODEX wizard software, select Microscope Pre-Check. Once it passes assessment, Start the run
?. [Microscope set up start acquisition]
Leave the microscope window open and on full view. Failure to do so may result in the driver software shutting down.
?. [Microscope set up start acquisition]
When run is complete, confirm there are no errors in the error log.
?. [Microscope set up start acquisition]
Close the microscope software and power off the microscope.