TITLE: CODEX Oligo-labeled Antibody Conjugation
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 href="http://akoyabio.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Akoya Biosciences</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Antibody Disulfide Reduction Reaction]
Retrieve one MWCO filter column for each antibody to be conjugated.  Block nonspecific antibody binding to the MWCO filter columns by adding 500ul Filter Blocking Solution to the top of each column and spinning down at 12,000g for 2 minutes.
0 Room temperature
?. [Antibody Disulfide Reduction Reaction]
Aspirate all liquid from the top of each MWCO filter column and discard flow-through in the collection tube.  Exercise caution and do not scrape and damage the filter with the aspirator tip.
?. [Antibody Disulfide Reduction Reaction]
Measure concentration of stock purified antibody by nanodrop with pre-set IgG settings after blanking against the buffer they are suspended in, usually PBS.  Calculate the volume needed for 50ug of antibody based on the nanodrop measurement.
?. [Antibody Disulfide Reduction Reaction]
Add 50ug of antibody to the top of each MWCO filter column and spin down at 12,000g for 8 minutes.  The resulting solution should be 50ug of antibody concentrated into approximately 25ul.
0 Room temperature
?. [Antibody Disulfide Reduction Reaction]
Discard flow-through.
?. [Antibody Disulfide Reduction Reaction]
Prepare Antibody Reduction Master Mix by combining Antibody Reduction Solution 1 with Antibody Reduction Solution 2.  20ul of Antibody Reduction Solution 1 is needed for every 3 antibodies to conjugate.20ul of Antibody Reduction Solution 1 is mixed with 825ul of Antibody Reduction Solution 2.
?. [Antibody Disulfide Reduction Reaction]
Add 260ul Antibody Reduction Master Mix to the top of each MWCO filter column.  Gently pipet up and down to mix reagent with antibody.  Incubate at RT for 30 minutes.
0 Room temperature
?. [Antibody Disulfide Reduction Reaction]
After 30 minutes spin down the MWCO filter columns at 12,000g for 8 minutes.
0 Room temperature
?. [Antibody Disulfide Reduction Reaction]
Discard flow-through.
?. [Antibody Conjugation Reaction]
Add 450ul Antibody Conjugation Solution to the top of each MWCO filter column and spin down at 12,000g for 8 minutes.  During this spin, prepare the antibody tags.
0 Room temperature
?. [Antibody Conjugation Reaction]
The antibody tag preparation is time sensitive and must be done immediately prior to use.  The antibody tag aliquots are to be used once for every 50ug antibody conjugation reaction.
?. [Antibody Conjugation Reaction]
Pipet 220ul Antibody Conjugation Solution into each antibody tag aliquot.  Ensure that the aliquot is dissolved with gentle pipetting.
?. [Antibody Conjugation Reaction]
After the spin in step 10 is completed, discard flow-through and add the dissolved antibody tag solutions from step 12 into each corresponding MWCO filter column.  Pipet up and down gently to mix the reagents.
?. [Antibody Conjugation Reaction]
Close the MWCO filter column lids and incubate the conjugation reaction at RT for 2 hours.
0 Room temperature
?. [Antibody Conjugation Reaction]
After 2 hours, spin down the MWCO filter columns at 12,000g for 8 minutes.
0 Room temperature
?. [Antibody Conjugation Reaction]
Discard flow-through.
?. [Antibody Purification and Storage]
Add 450ul Antibody Purification Solution to each MWCO filter column and spin down at 12,000g for 8 minutes.Discard flow-through.
0 Room temperature
?. [Antibody Purification and Storage]
Add another 450ul Antibody Purification Solution to each MWCO filter column and spin down at 12,000g for 8 minutes.Discard flow-through.
0 Room temperature
?. [Antibody Purification and Storage]
Add another 450ul Antibody Purification Solution to each MWCO filter column and spin down at 12,000g for 8 minutes.Discard flow-through.
0 Room temperature
?. [Antibody Purification and Storage]
Add 100ul Antibody Storage Solution to the top of each MWCO filter column.  Gently pipet up and down 10 or more times and wash the sides of the filters in the column.  50ug antibody should be dissolved in 100ul Antibody Storage Solution.  If the conjugation scale is larger than 50ug, add more Antibody Storage Solution according to this ratio.
?. [Antibody Purification and Storage]
Invert the filters into a fresh collection tube.  Spin down at 3000g for 2 minutes.  KEEP THE COLLECTED SOLUTION.
?. [Antibody Purification and Storage]
Pipet the conjugated antibody solutions into sterile screw-top tubes and store for one year at  .
4 °C