TITLE: Tissue Cyclic Immunofluorescence (t-CyCIF)_PCA2024_spacer
AUTHORS: Jia Ren Lin, Benjamin Izar, Zoltan Maliga, Yu-An Chen, Giorgio Gaglia, Ziming Du, Clarence Yapp, Shaolin Mei, Sandro Santagata, Peter Sorger

[DESCRIPTION]
The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immunofluorescence imaging of specimens mounted on glass slides.  t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation.  t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.

Key resources, reagents, and software are listed at the HMS LINCS Center Publication Page http://lincs.hms.harvard.edu/lin-elife-2018/ (RRID:SCR_016370). This page provides links to an OMERO image database from which individual images can be obtained; stitched and registered image panels can be obtained at www.cycif.org (RRID:SCR_016267) and a video illustrating the t-CyCIF method can be found at https://vimeo.com/269885646.

This protocol is used in the following manuscripts:  
Lin J-R, Izar B, Wang S, Yapp C, Mei S, Shah P, Santagata S, Sorger PK. (2018). Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. eLife. PMID: 29993362 
Du Z, Lin JR, Rashid R, Maliga Z, Wang S, Aster J, Izar B, Sorger PK, Santagata S. (2019). Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging. Nature Protocols. PMID: 31534232

The original t-CyCIF protocol can be found at dx.doi.org/10.17504/protocols.io.rpxd5pn. 

[BEFORE_START]
Note that t-CyCIF is optimized for FFPE specimens, which must first undergo dewaxing and antigen retrieval to expose antigenic sites for antibody binding. An example of an automated version of this process can be found here: dx.doi.org/10.17504/protocols.io.4zpgx5n.

[STEPS]
SECTION: Pre-Staining and Background Determination
1. Pre-staining and Background Determination takes approximately 16-24 hours.

Make fluorophore bleaching solution. Combine 165 mL 1X PBS, 30 mL 30% (wt/vol) H2O2, and 4 mL 1 Molarity (M) NaOH. The final working concentration is 4.5% (wt/vol) H2O2 and 20 millimolar (mM) NaOH in PBS. 199 mL of bleaching solution is enough for 30 standard slides.

CRITICAL STEP Fluorophore bleaching solution should be prepared immediately before use.
SECTION: Pre-Staining and Background Determination
2. Place slides flat in a plastic transparent container with the tissue facing up, and then gently pour fluorophore bleaching solution into the container to completely cover tissue. Sandwich the container between two LED light panels (one above and one below) at Room temperature for 2x 30 min.

CRITICAL STEP The pre-bleaching step is critical for reducing autofluorescence in the tissue and to inactivate the fluorophores of the secondary antibody from the pre-staining step. 
CRITICAL STEP Light sources that produce excessive heat can damage tissues. LED light sources are therefore preferable, and large flat LED panels are now readily available at low cost (see “Materials” for our preferred light panel).
CRITICAL STEP Completely immerse the tissue sections in fluorophore bleaching solution. During the subsequent bleaching process, bubbles will appear and gradually increase in size and number. This indicates that the oxidation reaction is proceeding as expected.
SECTION: Pre-Staining and Background Determination
3. Wash slides 4 times with 1X PBS at Room temperature for at least 5 min per wash. Slides can be placed into a slide rack and lowered into a staining dish of PBS.
SECTION: Pre-Staining and Background Determination
4. Place slides in the slide tray, completely cover tissues with the secondary antibody solution used in the pre-staining procedure with added Hoechst solution (2.5 µg/ml) and incubate in the dark at 4 °C overnight to block tissues from non-specific binding. 

CRITICAL STEP Place damp paper towels in the slide tray to maintain humidity and prevent evaporation of the antibody solution.
CRITICAL STEP Do not use a hydrophobic barrier pen on the slides, as we have found that this adversely affects results. 
CRITICAL STEP Be careful not to scratch the tissue with pipette tip when applying the antibody solution.
SECTION: Pre-Staining and Background Determination
7. Bleach slides at least 2x 30 min at Room temperature as previously described.
SECTION: Pre-Staining and Background Determination
8. Wash the slides 4 times with 1X PBS at Room temperature for 5 min per wash. 

CRITICAL STEP ensure PBS covers entire slide including label region to ensure all bleaching solution is removed from slides.
PAUSE POINT Slides may be stored in 1X PBS at 4 °C for several days. Avoid letting tissue dry; ensure entire tissue is covered in 1X PBS.
SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
9. The first round of t-CyCIF takes approximately 16-24 hours.

Dilute up to three unconjugated primary antibodies from different species to the appropriate concentration in    containing Hoechst 33342 (2.5 µg/ml), cover tissue, and incubate in the dark at 4 °C overnight. 

CRITICAL STEP Unconjugated antibodies used in CyCIF are limited by unique secondary antibody and flourophore combinations available; we limit indirect IF to first two cycles).The optimal dilution for primary antibodies must be optimized empirically; we usually test across a range of dilutions starting from 1:100, guided by manufacturer’s recommendations. The times listed for antibody incubation can be adjusted empirically; we use long incubations at 4 °C for convenience. (See Lin et al., 2018 and https://www.cycif.org/ (RRID:SCR_016267) for information on increasing the throughput of t-CyCIF experiments.)
SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
10. Wash slides 2 times with 1X PBS at Room temperature for 10 min per wash. The longer slides are washed/rinsed minimizes unbound antibody artifacts.

SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
11. Cover the tissue with secondary antibodies diluted in SuperBlock Blocking Buffer containing Hoechst  (2.5 µg/ml) . Incubate in the dark at Room temperature for 60 min.
SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
12. Wash slides 3 times with 1X PBS at Room temperature for 30 min per wash. 

SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
13. Mount coverslips on slides with 200 µL of 50% glycerol in PBS and image as described. 

CRITICAL STEP Use a saved ROI in the imaging software so that the exact same region of tissue is imaged for every cycle of t-CyCIF. 
TROUBLESHOOTING Weak signal. Possible reason: Low signal can result because of low level antigen expression. Direct immunofluorescence using conjugated antibodies does not provide the signal amplification that can be generated in indirect immunofluorescence. Solution: Increase the exposure time while acquiring image; increase the antibody concentration during staining step; use the corresponding unconjugated antibodies in the first round instead of the conjugated antibody to see if signal amplification from indirect immunofluorescence improves signal; if necessary, find an alternative antibody. 
TROUBLESHOOTING Saturating signal. Possible reason: Abundant antigen in sample or excessive amount of antibody. Solution: Decrease the antibody concentration used during the staining steps; decrease the incubation time of the sample with antibody; decrease the exposure time during image acquisition.
SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
14. After imaging, remove the coverslips as described above and wash the slides 2 times with 1X PBS at Room temperature for 3 min per wash.
SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
15. Perform fluorophore bleaching for 2x  30 min at Room temperature as described. 
SECTION: First (AND/OR second) Round of t-CyCIF (Indirect IF)
16. Wash slides 4 times with 1X PBS at Room temperature for 5 minper wash. 

CRITICAL STEP Wash slides thoroughly to remove fluorophore bleaching solution, since carry-over can adversely affect subsequent t-CyCIF cycles.
PAUSE POINT Slides may be stored in 1X PBS at 4 °C for several days. Make sure the entire tissue is covered in 1X PBS. Otherwise, the tissue may become dry and yield poor staining results. 
SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
17. Subsequent cycles of t-CyCIF use antibodies with directly conjugated fluorophores and each takes approximately 16-24 hours each. The maximum number of cycles for t-CyCIF is determined by how long the tissue structure is preserved between cycles. We are able to perform >10 cycles for most tissue types.

Dilute up to three conjugated antibodies conjugated with different fluorophores in SuperBlock® (PBS) Blocking Buffer containing Hoechst  (2.5 µg/ml). Cover tissue with antibody solution and incubate in the dark moisturized container at 4 °C overnight. 
CRITICAL STEP We use Alexa Fluor 488-, Alexa Fluor 555-, and Alexa Fluor 647- conjugated primary antibodies. Dilution is optimized empirically starting from 1:100 (vol:vol). 
CRITICAL STEP  Alexa Fluor 546-, Alexa Fluor 568-, and Alexa Fluor 594- conjugated secondary antibodies are difficult to bleach; Avoid use. 
SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
18. Wash the slides 3 times with 1X PBS at Room temperature for 30 min per wash.
SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
19. Mount coverslips with 200 µL of 70% (vol:vol) glycerol in PBS and image the saved ROI with the RareCyte CyteFinder as described.
SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
20. Remove the coverslips as described.
SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
21. Wash the slides 2 times with 1X PBS at Room temperature for 3 min per wash. 

SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
22. Bleach slides for 2x  30 min at Room temperature as described.
SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
23. Wash the slides 4 times with 1X PBS at Room temperature for 3 min per wash.
SECTION: Subsequent Cycles of t-CyCIF (Direct IF)
24. Start next t-CyCIF cycle: repeat steps 16-22  for each additional cycle.

TROUBLESHOOTING Blurry images.
TROUBLESHOOTING Weak signal.
TROUBLESHOOTING Saturating signal.
TROUBLESHOOTING Cell loss. Possible reason: Difficult tissue type (very low cell density). Insufficient tissue fixation. Damage from t-CyCIF procedure (e.g. rough handling of samples during washing). Solution: Check pre-analytical variables. Cautious handling of samples during application of antibodies and washing steps as well as during manipulation of coverslips. 
TROUBLESHOOTING Signal present after fluorophore bleaching step. Possible reason: Insufficient fluorophore inactivation. Solution: Avoid Alexa Fluor 546-, Alexa Fluor 568-, and Alexa Fluor 594- conjugated antibodies because they are difficult to inactivate; dilute conjugated antibodies further; extend fluorophore bleaching time; check that light is hitting the sample. 
SECTION: Pre-Staining and Background Determination
6. OPTIONAL Image background/autofluorescence
SECTION: Pre-Staining and Background Determination
6.1. Mount coverslips onto slides with 200 µLof 50% (vol:vol) glycerol in 1X PBS to prevent dehydration during imaging. Carefully position coverslips over the center of each slide and lower slowly onto the slide to avoid producing bubbles between the coverslip and to prevent scratching tissues. Do not allow coverslip to overhang the edge of the slide. Dry excess liquid by gently pressing the long edges of the slide against a paper towel. 

CRITICAL STEP Wet-mounting and positioning coverslips takes some practice that should be undertaken initially using non-precious specimens.
SECTION: Pre-Staining and Background Determination
6.2. Image background staining/autofluorescence using ex/em filters that will be used in the rest of the experiment. 

SECTION: Pre-Staining and Background Determination
6.3. Place slides in 1x PBS pre-warmed to 42°C until coverslips fall off the tissue - up to 1h.  Do not disturb the tissue spacer - it will remain on the slide through the remaining steps of the experiment.

SECTION: Pre-Staining and Background Determination
5. Prepare slides for cyclic imaging.
SECTION: Pre-Staining and Background Determination
5.1. Wash slides 4 times with 1X PBS at Room temperature for at least 5 min per wash. 
SECTION: Pre-Staining and Background Determination
5.2. Using a lint-free wipe, remove excess PBS from around the tissue. Carefully apply self-adhesive    onto the slide surrounding the tissue. If not imaging background, place slides in PBS  while making the fresh bleach solution for used in step 7.