Multiplexed IHC Staining with Primary Antibodies Conjugated to Fluorophores

The following protocol provides a step-by-step guide for performing multiplexed staining of a tissue sample using a combination of primary antibodies linked to fluorophores that target specific markers. We recommend using primary antibodies conjugated to fluorophores for multiplexed imaging experiments for medium to highly expressed markers. Additionally, this protocol includes instructions on how to create single-color controls that are essential for spectral unmixing of a multiplexed sample containing fluorophores with emission spectra that highly overlap.

• Primary antibodies to your targets specific for IHC
• Xylene (Cat. No. 396931000) (FFPE only)
• 100% Ethanol (Cat. No. T038181000) (FFPE only)
• Phosphate buffered saline (10X) pH 7.4, RNase-free (Cat. No. AM9624)
• Tissue Tek Staining Dishes (3) (Cat. No. NC9507198)
• Tissue Tek Clearing Agent Dishes (2) (Cat. No. NC9012500)
• Tween™ 20 (Cat. No. 85113)
• Mounting medium
  • ProLong Glass Antifade Mountant (Cat. No. P36984)
  • SlowFade Glass Soft-set Antifade Mountant (Cat. No. S36917)

• ReadyProbes Hydrophobic Barrier Pap Pen, (Cat. No. R3777)
• Antigen Retrieval Reagents:
  • Citrate Buffer (pH 6.0), Concentrate (Cat. No. 005000) or
  • eBioscience IHC Antigen Retrieval Solution - Low pH (10X) (Cat. No. 00-4955-58) or
  • eBioscience IHC Antigen Retrieval Solution - High pH (10X) (Cat. No. 00-4956-58)
• Hydrogen peroxide, 30% w/v (Example: Cat. No. H325-4)
• Sodium Hydroxide Solution (50% w/w/Certified) (Example: Cat. No. SS254500)
• NucBlue Fixed Cell ReadyProbes Reagent (Cat. No. R37606)

• Make sure to prepare enough tissue samples for the multiplex sample and the necessary single-color control samples.
• Do not let the tissue sample dry out. Dried out tissue will result in incorrect or no signal. Ensure that there is enough solution to completely cover the tissue during incubation and wash steps. We recommend using a humidified chamber (for example, a covered box with damp paper towel).
• Crisp staining results require optimizing primary antibody dilution.

The following negative and single-color controls are required for spectral unmixing of the multiplex. Please prepare each of the controls including:

We strongly recommend you optimize the primary antibody before doing the multiplex staining. Stain tissues with a serial dilution including 1:10, 1:50, 1:100, 1:500 of the primary antibody conjugated to a fluorophore.

Deparaffinize the tissue using a standard deparaffinization rehydration protocol.

Perform heat-induced epitope retrieval (HIER) using either Citrate Buffer (pH 6.0) or EDTA (pH 9) using a microwave or pressure cooker according to standard antigen retrieval protocols.
Note: An automated slide stainer can be used for the dewaxing/deparaffinization and HIER steps.

1. Depressurize and crack pressure cooker lid for 5 minutes to allow sample cooling.
2. Remove the slide rack with forceps, submerge it into a clear staining dish containing 200 mL of ddH₂O, and wash for 1 minute with frequent agitation.
3. Repeat the wash one more time with fresh ddH₂O.
4. Transfer the slide rack to 1X PBS.

Note: Do not let slides dry out from this point forward.
Note: Samples can be stored covered in 1X PBS at RT overnight.

1. Draw hydrophobic barrier with pen.
2. Allow barrier to dry at RT for 2-3 minutes.
3. Prepare 0.1% Triton/PBS
4. Remove each slide and flick excess 1X PBS, without completely drying out the sections.
5. Place the slides face up on a flat surface and immediately add 400 µL of the working permeabilization solution for 30 minutes at RT.
6. Decant the working perm buffer from the slides, and wash slides in 1X PBS thoroughly by shaking up and down for 1 minute.
7. Repeat wash with fresh 1X PBS, three times.

Perform autofluorescence reduction with white light prior to labeling (method described in Nat Cancer. 2023 Jul;4(7):1036-1052).

Prepare reagents:

Sodium hydroxide (NaOH) stock solution

Working autofluorescence solution

Note: Final concentration should be 24 mM NaOH and 4.5% H₂O₂ in PBS.

1. Rinse samples with 1X PBS at room temperature.
2. Place slides in a clear container covered with the working autofluorescence solution (4.5% hydrogen peroxide and 24 mM NaOH in PBS).
3. Illuminate with white light for 30 min.

Note: We do not recommend using the UV light as it can destroy certain antigens.

4. Wash 3x in 1X PBST (0.05% Tween™ 20)

1. Block with 3% BSA for 1 hour at RT.

1. Create single-color controls and unstained control. Unstained control must be used with every sample block.
2. Create your multiplex master mix by diluting in each of your primary antibody conjugates to desired concentration in 3% BSA blocking buffer. We recommend having a final dilution of 1:10 (final concentration 0.02 mg/mL) if you did not optimize the dilution for the multiplex staining.
3. Add to sample and incubate 1 hour at RT in a humidified chamber in the dark.
   
   Note: Staining can be incubated overnight at 4°C. This may increase both intensity of staining or background.
   
   
4. Wash 3x with 1X PBST (0.05% Tween™ 20)
5. Add nuclear counter stain such as NucBlue stain or DAPI.
6. Wash 3x with 1X PBST (0.05% Tween™ 20). Do not soak samples in 1X PBST longer than 30 minutes.
7. Mount the coverslips using a mountant with antifade properties:

• ProLong Glass Antifade Mountant (Cat. No. P36984) is recommend for hard-set mounting and long-term archiving of tissue samples.
• SlowFade Glass Soft-set Antifade Mountant (Cat. No. S36917) is recommended for soft-set mounting if coverslip removal is required for tissue samples.

Note: For optimal results, follow the instructions provided with the mountant.

8. Analyze the tissue using a compatible imaging instrument.

Lin JR, Chen YA, Campton D, Cooper J, Coy S, Yapp C, Tefft JB, McCarty E, Ligon KL, Rodig SJ, Reese S, George T, Santagata S, Sorger PK. High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers. Nat Cancer. 2023 Jul;4(7):1036-1052. doi: 10.1038/s43018-023-00576-1. PMID: 37349501