TITLE: Antibody Conjugation and CODEX Multiplexed Immunofluorescence of Fresh Frozen Tissue
AUTHORS: Elizabeth Neumann, Carrie  Romer, Nathan Heath Patterson, Jamie Allen, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes antibody conjugation, tissue preparation, and microscopy for CODEX multiplexed immunofluorescence analysis of fresh frozen tissue.</div></div>

[STEPS]
?. [Antibody Conjugation for Custom Antibody Markers]
Start with purified antibodies (no BSA, azides, glycerol, etc.). If BSA is present, remove with ab173231.
?. [Antibody Conjugation for Custom Antibody Markers]
Block 50 kDa molecular weight cut off filters with   of filter blocking solution.
500 µl
?. [Antibody Conjugation for Custom Antibody Markers]
Centrifuge at  for  .
Centrifuge: 12000 34
?. [Antibody Conjugation for Custom Antibody Markers]
Dilute 50 µg of each antibody into  of PBS and filter with 50 MW cut off filters.
100 µl
?. [Antibody Conjugation for Custom Antibody Markers]
Centrifuge at  for .
Centrifuge: 12000 34
?. [Antibody Conjugation for Custom Antibody Markers]
Reduce antibodies with the reduction mixture for 20-25 minutes at ~.
20 °C
25 minutes of reduction is suitable for many antibodies but may need to be optimized (15 min-40 min) depending on the primary antibody used. In general, shorter reduction times are more appropriate than longer times.
?. [Antibody Conjugation for Custom Antibody Markers]
Centrifuge the reduced antibodies at  for  and add buffer solution.
Centrifuge: 12000 34
?. [Antibody Conjugation for Custom Antibody Markers]
Rehydrate oligonucleotide barcodes in    nuclease free water and further dilute with    of conjugation solution.
10 µl
210 µl
Nuclease free water must be used, otherwise the oligonucleotide barcodes will be degraded.
?. [Antibody Conjugation for Custom Antibody Markers]
Respective barcode solutions are added to the reduced primary antibodies and incubated for  at ~
20 °C
?. [Antibody Conjugation for Custom Antibody Markers]
*Purify the solution by buffer exchanging with   purification solution. Centrifuge at   for  . Discard flow-through.
450 µl
Centrifuge: 12000 34
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Remove fresh frozen tissue from   and place inside a desiccator for .
-80 °C
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Submerge tissues in excess acetone for .
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Rehydrate tissues in hydration buffer for   inside a humidity chamber.
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Fix the tissues for  in 1.6% paraformaldehyde in hydration buffer.
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Submerge tissues in excess staining buffer for .
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Incubate tissues with a blocking buffer and primary antibody cocktail (1:200 antibody dilution) for  within a humidity chamber at ~ .
20 °C
Tissues can be incubated in primary antibody solutions overnight if placed inside a refrigerator.  Background may increase if longer incubation times are used but may be necessary for some antigen targets.
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Rinse samples with staining buffer and fix with 1.6% paraformaldehyde in storage buffer for .
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Rinse with excess PBS and incubate in cold methanol for .  Rinse samples with excess PBS again.
?. [Tissue Preparation for CODEX Multiplexed Analysis]
Fix the samples with fixative solution for  before being stored in storage buffer at .
4 °C
?. [Fluorescence Microscopy and CODEX Multiplexed Immunofluorescence]
Dilute antibodies in reporter solution to a 1:200 dilution with   of a  solution of DAPI or Hoechst 33342.
1 µl
Most secondary oligonucleotide barcode reporters function at a 1:200 dilution; however, this dilution may need to be optimized in select cases or with custom reagents.
?. [Fluorescence Microscopy and CODEX Multiplexed Immunofluorescence]
Fill respective bottles with nuclease free water, 1x CODEX buffer, and dimethyl sulfoxide.
?. [Fluorescence Microscopy and CODEX Multiplexed Immunofluorescence]
Mount a blank coverslip into the coverslip microfluidic chamber and flush the fluidic lines.
?. [Fluorescence Microscopy and CODEX Multiplexed Immunofluorescence]
Mount the prepared tissue sample and incubate with 1x CODEX buffer and  of   of DAPI for  and wash with the automated CODEX system.
1 µl
?. [Fluorescence Microscopy and CODEX Multiplexed Immunofluorescence]
Set up tiled regions covering the tissue and focus at the tissue surface.
?. [Fluorescence Microscopy and CODEX Multiplexed Immunofluorescence]
Perform automated microscopy using a Zeiss Axio equipped with a Colibri 7 LED light source and C13440 camera or other equivalent equipment.
Important parameters include a 10% tile overlap and a z-stack ranging from 11 to 20 tiles (depending on the tissue size).
?. [Antibody Conjugation for Custom Antibody Markers]
twice.
?. [Antibody Conjugation for Custom Antibody Markers]
Store in  antibody storage solution at  .
100 µl
4 °C