Introduction

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.

Materials

Optional materials


Procedure

Procedural guidelines and tips

  • 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.

Prepare controls

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

Single-color controls

Protocol

Single-color control: Make one for each primary antibody conjugate found in multiplex stainingDilute the primary antibody conjugate in 3% BSA blocking buffer to the same concentration as was used in the multiplex sample.
Add to sample and incubate 1 Hour at RT in a humidified chamber in the dark.
Wash 3x with 1X PBST (0.05% Tween™ 20)
Proceed to mounting step. Do not use any counter stain.
Nuclear counter stain only controlAdd nuclear counter stain such as NucBlue or DAPI at the same concentration as was used in the multiplex sample.
Wash 3x with 1X PBST (0.05% Tween™ 20).
Proceed to mounting step.
Unstained tissue sampleNo counter stain or any fluorophore conjugates.
Proceed to mounting step.

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.

Tissue preparation

Deparaffinize the tissue using a standard deparaffinization rehydration protocol.

Solution

Incubation time

Xylene5 minutes
Xylene5 minutes
1:1 solution of Xylene : 100% EtOH5 minutes
100% EtOH3 minutes
95% EtOH3 minutes
85% EtOH3 minutes
75% EtOH3 minutes
50% EtOH3 minutes
dH2ORinse
1X PBS5 minutes
1X PBS5 minutes
1X PBS5 minutes

Antigen retrieval

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.

Permeabilization

  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.

White light photobleached (autofluorescence treatment—optional)

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

Reagent

Volume

NaOH solution (50% w/w)0.5 mL
Deionized water9.5 mL


Working autofluorescence solution

Reagent

Volume

1M NaOH Stock2.4 mL
H2O2 (30% w/v)4.5 mL
PBS93.1 mL

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)

Block samples for non-specific binding.

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

Multiplex labeling with primary antibodies conjugated to fluorophores.

  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:

    8. Analyze the tissue using a compatible imaging instrument.


    References

    For Research Use Only. Not for use in diagnostic procedures.