IHC-IF Protocol - Multiplex

This protocol describes Multiplex IHC staining using IF detection. 

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Multiplex immunofluorescence allows the simultaneous detection of several target proteins in the same cell. Primary antibodies should ideally be from different species, but in some cases, it is possible to use antibodies raised in the same species. In the latter case, it is possible to use e.g. primary antibodies of different isotypes, which can then be visualized using subtype-specific secondary antibodies.

The secondary antibodies should be from the same species (to prevent cross-reactivity). Many companies have special antibodies with minimal cross-reactivity developed especially for co-localization studies. Moreover, several control experiments must be performed to ensure the specificity of co-localization of different targets (see below).

Materials needed

• Cryo- or paraffin-embedded sections mounted on SuperFrost slides
• Target retrieval solutions pH6 or pH9 (if needed)
• Incubation chamber with wet Wettex stripes
• DAKO Cytomation pen
• 1xPBS
• TBS or TNT wash buffer
• Primary and secondary antibodies diluted in 1x PBS, containing 0.3% Triton, 0.01% NaAzide, 0.02% Bacitracin and BSA
• Fluorophore-conjugated secondary antibodies for regular immunofluorescence
• Coverslips
• Eppendorf tubes for dilution of antibodies
• Pipettes and pipette-tips

Primary antibody preparation

• Pre-incubate primary antibody with BSA (0.5%) prior to application to the tissue.
• Dilute primary antibody in antibody diluent to a working concentration.

Cryosections

Perfusion-fixed: 10-30% sucrose cryoprotected sections cut in a microtome at 14 μm, thaw-mounted on Super Frost slides. Dry sections on slides for an additional 2 h at room temperature (RT). Rehydrate in 1xPBS 2x15 min, or proceed with antigen retrieval.

Snap-frozen: Sections cut in a microtome at 14 μm, thaw-mounted on Super-Frost slides and dried for 2 h. Pre-fix sections in ice-cold paraformaldehyde (4% PFA) for 20 min (put frozen sections directly into 4% PFA, do not allow thawing). Rinse in 1xPBS 15 min.

Paraffin-embedded tissue

Perfusion- or immersion-fixed paraffin-embedded sections cut at 4 μm thick, mounted on Super Frost slides, dried at 50°C overnight.

Antigen retrieval for cryosections (optional)

If the target requires antigen retrieval, standard target retrieval solutions can be used, though the temperature and time should be reduced.

Heat-Induced Epitope Retrieval (HIER)

1. HIER buffer pH 6, a modified citrate buffer, 60-70°C, 10 min.
2. HEIR buffer pH 9, a Tris/EDTA buffer, 60-70°C, 10 min.

Antigen retrieval for paraffin sections

Heat-Induced Epitope Retrieval
(HIER)

1. HIER buffer pH 6, a modified citrate buffer, 97°C, 20 min.
2. HIER buffer pH 9, a Tris/EDTA buffer, 97°C, 20 min.

Cool down with ddH2O, rinse in 1x wash buffer for 15 min, and proceed with IHC.

If antigen retrieval is not required

1. Deparaffinize sections in xylene (2x5 min).
2. Rehydrate sections: 100% EtOH 2x3 min, 95% EtOH 2x3 min, 70% EtOH 2x3 min, rinse in ddH20.
3. Proceed with the IHC procedure.

NOTE: The specified working dilutions of the primary antibodies are to be considered as a guideline only. Optimal dilutions must be determined by the user. If primary antibodies are of different isotypes (IgG1, IgG2a, IgG2b), they can be mixed together. 

IHC procedure

1. Draw a circle around each section with DAKO Cytomation pen.
2. Rinse slides in 1x wash buffer for 15 min
3. Blocking endogenous peroxidase
4. Cryosections: Put slides in 0.03-0.1% H2O2 in 1xPBS for 5 min at RT to quench endogenous peroxidase activity
5. Paraffin sections: Put slides in 0.3% H2O2 in 1xPBS for 15 min at RT to quench endogenous peroxidase activity
6. Rinse in PBS for 15 min at RT
7. Block sections in 2% normal serum of the secondary antibody host, in PBS 30 min at RT (optional)
8. Add primary antibodies (diluted in 1x PBS, containing 0.3% Triton, 0.01% NaAzide, 0.02% Bacitracin), approx. 180μl/slide. 
9. Incubate in a humidified chamber overnight at 4ºC.

Day 2

IHC-IF:

1. Rinse slides in 1x wash buffer for 3x5 min at RT
2. Incubate with secondary antibodies conjugated with fluorophores (1:80 -1:800 in PBS), 30 min at 37ºC or 1 h at RT in the dark. Isotype-specific secondary antibodies can be mixed in the same incubation solution.
3. Wash in wash buffer for 2x10 min at RT in the dark.
4. Mount in anti-fading solution (e.g. ProLong Gold), and let cure for 24 h at 4ºC
   Ready for microscopy/scanning
5. Store at 4ºC.

Control protocols

Adsorption

For each primary antibody, an adsorption control should be done on single-stained sections:

Incubate primary antibody (in working dilution which will be used on the section next day) with corresponding peptide 10-6M overnight at 4ºC in an Eppendorf tube pre-coated with Sigmacote (trimetylcylan).

Apply absorbed antibody on sections as in normal protocol Day I for overnight incubation, and proceed as for IHC Day 2.

Tissue-IgG interactions

Omitting primary antibodies should result in blank sections.

Single-stain control

The immunoreactivity observed on slides with co-labeling should be identical to the expression profile on adjacent single-stained sections.

Co-localization controls

(Exemplified by double-labeling with Anti-X IgG1 and Anti-Y IgG2a).

I. Positive signal only in the appropriate channel
Labeled cells should only be observed in the corresponding channel (and not in other channels).

Slide 1 MAb Anti-X IgG1 -> Goat Anti-Mo-IgG1* Alexa 488 -> Only a green signal should be observed.
Slide 2 MAb Anti-Y IgG2a-> Goat Anti-Mo-IgG2a* Alexa 594 -> Only a red signal should be observed.

II. The primary antibody does not react with non-corresponding secondary antibody

No signal should be observed (blank sections)

Slide 3 MAb Anti-X IgG1 -> Goat Anti-Mo-IgG2a* Alexa 594 -> No signal
Slide 4 MAb Anti-Y IgG2a -> Goat Anti-Mo-IgG1* Alexa 488 -> No signal

III. Control of secondary antibody specificity

Secondary antibodies should only react with proper primary antibodies. If staining is observed in the non-corresponding channel, secondary antibodies interact with each other.

Slide 5 MAb Anti-X IgG1 -> Goat Anti-Mo-IgG1* Alexa 488 + Goat Anti-Mo-IgG2a* Alexa 594 -> Only green signal should be observed
Slide 6 MAb Anti-Y IgG2a -> Goat Anti-Mo-IgG1* Alexa 488 + Goat Anti-Mo-IgG2a* Alexa 594 -> Only red signal should be observed