Multiplexing and Spectral Microscopy Protocol

Overview

Introduction of Multiplexing and Spectral Microscopy Protocol

Immunohistochemistry (IHC) has long been employed to identify structural changes associated with disease or tissue injury, and has become an invaluable tool in diagnostic pathology and research, however a lack of distinct spectral profiles for chromogens and fluorophores used in IHC limits any investigation of protein distribution within a tissue section to one or two proteins. In recent years, protein arrays, RNA sequencing, and other high throughput molecular analyses have offered significant insights into disease biology. However, while these techniques provide important information on a tissue's overall expression patterns, they fail to discern the spatial distribution of proteins, limiting our understanding of how different cells and tissues interact during disease. In addition, the more our understanding of an innumerable number of signaling pathways increases, their complexity becomes evident and thus the need to simultaneously examine their multiple proteins, their interactions, and their molecular activities. Also, in the fields of immunology and cancer, the abundant number and phenotypes of cells of the tumor microenvironment, and their intricate interactions with normal host and neoplastic cells make conventional immunohistochemistry and even double labeling insufficient to unravel. The emergence of spectral microscopy offers an imaging modality capable of simultaneously visualizing up to eight proteins, providing additional insight into the phenotypes, distributions, and coexpression patterns of proteins in normal and pathological tissues while maintaining tissue architecture.

Spectral microscopy combines three techniques, microscopy, spectroscopy, and image analysis using linear unmixing, to separate the spectral signatures of chromogens or fluorophores while retaining high resolution images of tissue specimens. Similar to z-stack and time-lapse imaging, spectral microscopy uses a threedimensional dataset of images taken from the same field of view at different wavelengths to distinguish individual chromogens or fluorophores from each other. Each pixel in a given image has a value (x, y, λ) which denotes its physical location in the two-dimensional image at a given wavelength, with spectral resolution dependent on the intervals of λ. Reconstruction of a single-channel fluorescence image is then based on comparisons between the spectral signature of a given pixel (x, y) across the spectrum of λ and the corresponding spectral signals single-labeled reference samples imaged under the same parameters as the experimental sample. Linear unmixing relies on the principle that fluorescent emissions combine in a linear manner. By determining the intensity of each fluorescent signal at a given location based on these reference images and using the assumption that the sum of relative contributions of all fluorophores will be equate to 100%, linear unmixing separates each signal to distinguish individual fluorescent markers. Because tissue autofluorescence just represents another spectral signal, exogenous labels can be distinguished from tissue autofluorescence using linear unmixing, aiding in the visual detection of protein colocalization. Spectral unmixing is similarly applied to the separation of chromogenic spectra in brightfield spectral microscopy, though larger spectral signatures and reliance on detecting chromogen absorption rather than a linear relationship between signals limits the number of chromogens able to be distinguished and is heavily reliant on limiting background staining.

Optimized multiplexed immunohistochemistry (mIHC) protocols are critical for the success of spectral imaging. Simultaneous detection of multiple proteins within a single tissue section raises important considerations for antibody selection with regards to specificity and host species. While direct mIHC enables the use of multiple antibodies from the same host, it exhibits lower signal amplification than indirect IHC. Considering the potential for one highly expressed antigen to mask the signal of other antigens in spectral microscopy, indirect labeling holds key advantages over direct mIHC; however, traditional indirect mIHC requires primary antibodies to be raised in different host species. Recently, several protocols that enable indirect mIHC with antibodies raised in the same host species have been developed. These methods have greatly increased the ease with which scientists and pathologists may employ spectral microscopy.

Enzymatic Multiplexed Immunohistochemistry

Brightfield microscopy is a long-standing staple of pathology practice. By allowing for visualization of tissue architecture and protein distribution, it offers a key advantage over fluorescent microscopy; however, protein colocalization cannot be easily evaluated in conventional brightfield microscopy. Brightfield spectral microscopy can distinguish the spectral signals of up to four chromogens, eliminating the need for highly contrasting chromogens and the necessity to visualize targets in distinct cellular compartments. Key criteria must be met in enzymatic mIHC protocols for spectral microscopy, including high antibody specificity, low background staining, and careful considerations to prevent antibody crossreactivity during labeling.

Materials

Slide Preprocessing and Antigen Retrieval

Formalin-fixed, paraffin-embedded (FFPE) slides.
Oven.
Xylene.
Histological grade ethanol (prepared at 100%, 90%, and 70% concentrations in distilled water).
30% hydrogen peroxide
Methanol.
Phosphate buffered saline (PBS).
Citrate buffer pH 6.0.
Distilled water.

10. Humidified chamber (see Note 1).

Blocking

5% normal horse serum (for mouse monoclonal antibodies).
5% normal goat serum (for rabbit monoclonal antibodies).
0.1% PBS–BSA.

Primary Antibodies, Secondary Antibodies, and Chromogens

Primary antibodies (see Note 2).
HRP- and AP-conjugated secondary antibodies (see Note 3).
Avidin–biotin complex (ABC) (for biotinylated secondary antibodies).
Diamino-benzidine tetrahydrochloride (DAB).
Liquid Permanent Red (LPR) (see Note 4).
Vector Blue.
Vector VIP.
100 mM Tris–HCl, pH 8.5 buffer.

Counter-Staining and Mounting

Hematoxylin.
Acid alcohol.
Ammonia water.
Graded ethanol washes.
Xylene.
Xylene substitute.
Permount.
Glass coverslips.
InForm Advanced Image Analysis software.

Methods

Slide Preprocessing

Soften paraffin slides in the oven at 57–60°C for 40 min.
Dewax slides in xylene three times for 30 min each.
Rehydrate tissue in graded ethanol washes (100%, 90%, 70%, water) two times in each wash for 3 min per wash.
Quench endogenous peroxidase activity in 30% hydrogen peroxide in methanol for 20 min (40 mL 30% hydrogen peroxide + 160 mL methanol).
Rinse thoroughly and wash slides three times in PBS.
Heat-induced epitope retrieval (HIER):
a) Heat 200 mL citrate buffer pH 6.0 to 85°C.
b) Place slides in heated buffer and incubate in the oven at 95°C for 20 min.
c) Cool slides for 20 min.
Rinse and wash slides three times in PBS.

Two Primaries Raised in Different Species (Mouse–Rabbit Combination)

Block sections in 5% normal horse serum (for mouse monoclonal antibody) and 5% normal goat serum (for rabbit polyclonal antibody) prepared in 0.1% PBS–BSA for 2 h (see Note 5).
Incubate sections in a cocktail of both primary antibodies, diluted in 0.1% PBS–BSA to the desired concentration overnight at room temperature (see Note 6).
Rinse and wash slides three times in 1XPBS.
Incubate sections in a cocktail of both secondary antibodies (horse anti-mouse Ig/HRP and goat anti-rabbit Ig/AP) diluted to the desired concentration in 0.1% PBS–BSA for 1 h at room temperature.
If using biotinylated secondary antibodies (else, go to step 6):
a) Prepare avidin–biotin complex (ABC). Add two drops of avidin and two drops of biotin into 5 mL of 0.1% PBS– BSA. Let sit for 1 h before use.
b) Rinse thoroughly and wash slides three times in 1x PBS.
c) Apply ABC for 1 h at room temperature.
d) Continue with step 6.
Prepare LPR substrate. Add one drop of LPR Chromogen per 3 mL of LPR Substrate Buffer and mix well.
Prepare DAB substrate. Dissolve tablets (DAB first) in 5 mL distilled water.
Rinse and wash slides three times in 1XPBS.
Apply LPR to the sections for 10 min or less.
Rinse and wash slides in distilled water.
Apply DAB to the sections for 3 min or less.
Rinse and wash slides in tap water.
Perform a light nuclear counterstain (wash slides in tap water between steps):
a) Hematoxylin for 4–5 min.
b) Remove hematoxylin excess with 1 quick dip in acid alcohol.
c) Reblue slides with 5–6 dips in ammonia water.
Dry slides for 1 h at 60°C (see Note 7).
Clear slides in xylene for 5 min.
Mount with Permount.

Two Primaries Raised in the Same Species (Mouse–Mouse or Rabbit–Rabbit Combinations)

Block sections in 5% normal horse serum (for mouse monoclonal antibody) or 5% normal goat serum (for rabbit polyclonal antibody) prepared in 0.1% PBS–BSA for 2 h.
Incubate sections in primary antibody diluted in 0.1% PBS– BSA to the desired concentration overnight at room temperature.
Rinse and wash sections three times in 1XPBS.
Incubate sections in secondary antibody (conjugated to HRP for the first staining and AP for the second staining) diluted to the desired concentration in 0.1% PBS–BSA for 1 h at room temperature.
If using biotinylated secondary antibodies (else, go to step 6):
a) Prepare avidin–biotin complex (ABC). Add two drops of avidin and two drops of biotin into 5 mL of 0.1% PBS– BSA. Let sit for 1 h before use.
b) Rinse thoroughly and wash slides three times in 1XPBS.
c) Apply ABC for 1 h at room temperature.
d) Continue with step 6.
Prepare DAB substrate. Dissolve tablets (DAB first) in 5 mL distilled water.
Rinse and wash slides three times in 1XPBS.
Apply DAB to the sections for 3 min or less.
Rinse and wash slides in tap water.
PerformHIER to remove the first set of primary antibodies (see Note 8).
a) Heat 200 mL citrate buffer pH 6.0 to 85°C.
b) Place slides in heated buffer and incubate in the oven at 95°C for 20 min.
c) Cool slides for 20 min.
Repeat steps 1–5 for the second primary antibody and secondary antibody (conjugated to AP). If not using a biotinylated secondary antibody, skip step 5 and continue with step 11.
Prepare LPR substrate. Add one drop of LPR Chromogen per 3 mL of LPR Substrate Buffer and mix well.
Rinse and wash slides three times in 1XPBS.
Apply LPR to the sections for 10 min or less.
Rinse and wash slides in distilled water.
Perform a light nuclear counterstain (wash slides in tap water between steps):
a) Hematoxylin for 4–5 min.
b) Remove hematoxylin excess with 1 quick dip in acid alcohol.
c) Reblue slides with 5–6 dips in ammonia water.
Dry slides for 1 h at 60°C (see Note 7).
Clear slides in xylene for 5 min.
Mount with Permount.

Triple Labeling

Complete steps 1–12 of Two Primaries Raised in Different Species (Mouse–Rabbit Combination) or steps 1–15 of Two Primaries Raised in the Same Species (Mouse–Mouse or Rabbit–Rabbit Combinations) for the first two targets.
Perform HIER to strip primary and secondary antibodies from the tissue section.
a) Heat 200 mL citrate buffer pH 6.0 to 85°C.
b) Place slides in heated buffer and incubate in the oven at 95°C for 20 min.
c) Cool slides for 20 min.
Quench remaining peroxidase activity in 30% hydrogen peroxide in methanol for 20 min (40 mL 30% hydrogen peroxide + 160 mL methanol).
Incubate sections in primary antibody diluted in 0.1% PBS– BSA to the desired concentration overnight at room temperature.
Rinse and wash slides three times in 1XPBS.
Incubate sections in secondary antibody (conjugated to HRP) diluted to the desired concentration for 1 h at room temperature.
If using biotinylated secondary antibodies (else, go to step 8):
a) Prepare avidin–biotin complex (ABC). Add two drops of avidin and two drops of biotin into 5 mL of 0.1% PBS– BSA. Let sit for 1 h before use.
b) Rinse and wash slides three times in 1XPBS.
c) Apply ABC for 1 h at room temperature.
d) Continue with step 8.
Prepare Vector VIP. Add 3 drops of each Vector VIP reagent (Reagents 1, 2, 3, and 4) to 5 mL 1XPBS.
Rinse and wash slides three times in 1XPBS.
Apply Vector VIP to the sections for 10 min or less.
Rinse and wash slides in tap water for 5 min.
Perform a light nuclear counterstain (wash slides in tap water between steps):
a) Hematoxylin for 4–5 min.
b) Remove hematoxylin excess with 1 quick dip in acid alcohol.
c) Reblue slides with 5–6 dips in ammonia water.
Dry slides for 1 h at 60°C.
Clear slides in xylene for 5 min.
Mount with Permount.

Quadruple Labeling

Complete steps 1–12 of Two Primaries Raised in Different Species (Mouse–Rabbit Combination) or steps 1–15 of Two Primaries Raised in the Same Species (Mouse–Mouse or Rabbit–Rabbit Combinations) for the first two targets.
Perform HIER to strip primary and secondary antibodies from the tissue section.
a) Heat 200 mL citrate buffer pH 6.0 to 85°C.
b) Place slides in heated buffer and incubate in the oven at 95°C for 20 min.
c) Cool slides for 20 min.
Quench remaining peroxidase activity in 30% hydrogen peroxide in methanol for 20 min (40 mL 30% hydrogen peroxide + 160 mL methanol).
Incubate sections in primary antibody diluted in 0.1% PBS– BSA to the desired concentration overnight at room temperature.
Rinse and wash slides three times in 1XPBS.
Incubate sections in secondary antibody (conjugated to AP for the third staining and HRP for the fourth staining) diluted to the desired concentration for 1 h at room temperature.
If using biotinylated secondary antibodies (else, go to step 8):
a) Prepare avidin–biotin complex (ABC). Add two drops of avidin and two drops of biotin into 5 mL of 0.1% PBS– BSA. Let sit for 1 h before use.
b) Rinse and wash slides three times in 1XPBS.
c) Apply ABC for 1 h at room temperature.
d) Continue with step 8.
Prepare Vector Blue. Add 2 drops of each Vector Blue reagent (Reagents 1, 2, and 3) to 5 mL 100 mM Tris–HCl, pH 8.5 buffer.
Rinse and wash slides three times in 1XPBS.
Apply Vector Blue to the sections for 20 min.
Rinse and wash slides in 100 mM Tris–HCl, pH 8.5 buffer.
Rinse and wash slides in tap water.
Perform HIER to remove the third set of primary antibodies.
a) Heat 200 mL citrate buffer pH 6.0 to 85°C.
b) Place slides in heated buffer and incubate in the oven at 95°C for 20 min.
c) Cool slides for 20 min.
Repeat steps 4–7 for the second primary antibody and secondary antibody (conjugated to AP). If not using a biotinylated secondary antibody, skip step 7 and continue with step 15.
Prepare Vector VIP. Add 3 drops of each Vector VIP reagent (Reagents 1, 2, 3, and 4) to 5 mL 1XPBS.
Rinse and wash slides three times in 1XPBS.
Apply Vector VIP to the sections for 10 min or less.
Rinse and wash slides in tap water for 5 min.
Dry slides for 1 h at 60°C.
Clear slides in a xylene substitute (D-Limonenes, i.e., Histoclear) for 5 min (see Note 9).
Mount with Permount.

Notes

For optimal results, perform all incubation steps in a humidified chamber.
If possible, select primary antibodies raised in different species to eliminate the need for HIER. Mouse monoclonal and recombinant rabbit monoclonal antibodies yield improved results.
Secondary antibodies should be raised to match the primary antibody species (i.e., horse anti-mouse, goat anti-rabbit).
Any combination of chromogens can be used for brightfield enzymatic mIHC provided there is sufficient contrast between chromogenic precipitates.
If secondary antibodies are raised in a species besides horse or goat, normal serum raised in the same species as the secondary antibody should be used. Alternatively, a commercially available protein block may be used in place of normal serum.
Incubation times for the primary antibodies vary and should be optimized before mIHC.
If not using DAB and/or LPR, follow slide dehydration/ mounting protocols specific to the chromogens being used.
DAB covers binding sites on the primary and secondary antibodies. If DAB is used first, this can eliminate the need for HIER provided antibody concentrations have been optimized. For a more thorough review of antibody/chromogen combinations and alternate staining sequences for enzymatic mIHC, see van der Loos review.
Vector Blue is partially soluble in xylene, so clearing must be performed in a xylene substitute.

Fluorescent Multiplexed Immunohistochemistry

Figure 1. Multiplexing Examples.

Fluorescent mIHC offers a distinct advantage over enzymatic mIHC in its ability to detect additional targets. Several approaches have been utilized for fluorescent mIHC, including metal-based imaging mass cytometry, DNA barcoding, and tyramide signal amplification (TSA). One of the key elements of this modality is the microwave detachment of the previously incubated primary antibodies, which leaves the fluorophore attached to the antigen, preventing species cross-reactivity and allowing tremendous versatility and specificity to the procedure. While TSA multiplexing is currently limited to the detection of a maximum of six fluorophores, plus a nuclear counterstain (DAPI), it offers significant advantages regarding lower cost and ease of use compared to other methods.

Materials

Slide Preprocessing

Formalin-fixed, paraffin-embedded (FFPE) slides.
Oven.
Xylene.
Histological grade ethanol (prepared at 100%, 90%, and 70% concentrations in distilled water).
Distilled water.
10% neutral buffered formalin
Humidified chamber (see Note 1).

Microwave Treatment

1000 Wor higher microwave oven with 10+ adjustable power settings
10XPerkinElmer AR6 buffer (see Note 2)
Microwave-safe slide processing jars.
Milli-Q (ultrapure) water.
1XTBST.

Blocking and Antibodies

PerkinElmer Antibody Diluent/Block.
Hydrophobic barrier pen.
Primary antibodies (see Note 3).
Secondary Opal Polymer HRP Ms. + Rb.
Opal fluorophores (Opal 520, 540, 570, 620, 650, 690).
DMSO.
1XPlus Amplification Diluent.

Counterstain and Mounting

Spectral DAPI.
Aqueous fluorescence-safe mounting medium.
Glass coverslips.
Mantra Quantitative Pathology Imaging System.
InForm Advanced Image Analysis Software.

Methods

Slide Preprocessing

Soften paraffin slides in the oven at 57–60°C for 40 min.
Dewax slides in xylene three times for 30 min each.
Rehydrate tissue in graded ethanol washes (100%, 90%, 70%, water) two times in each wash for 3 min per wash.
Rinse slides in three times distilled water.
Fix slides in 10% neutral buffered formalin for 20 min.
Prepare 1XAR6 buffer. Dilute 10XAR6 buffer 1:10 in Milli-Q water.
Rinse slides two times in distilled water.
Rinse and wash slides two times in 1XAR6 buffer.
Transfer slides to a microwave-safe processing jar filled with 1XAR6 buffer.
Perform microwave treatment as follows:
a) 100% power until boiling point is reached (approximately 60 s).
b) 20% power for 15 min.
c) Cool slides at room temperature for 20 min.
Rinse and wash slides two times in distilled water.
Rinse and wash slides two times in TBST.

Labeling and Signal Generation for Seven-Color Manual

Surround the tissue section using a hydrophobic barrier pen.
Cover the tissue section with PerkinElmer Antibody Diluent/ Block and incubate for 10 min at room temperature.
Incubate sections in the first primary antibody diluted in PerkinElmer Antibody Diluent/Block to the desired concentration for 1 h at room temperature (see Note 4).
Rinse and wash slides three times in 1XTBST.
Incubate sections with Opal Polymer HRP Ms. + Rb secondary for 10 min at room temperature.
Prepare Opal Fluorophores. Reconstitute fluorophores with 75μL of DMSO. Prior to use, dilute each of the fluorophores 1:100 in Amplification Diluent.
Rinse and wash slides three times in 1XTBST.
Incubate sections with one Opal Fluorophore for 10 min at room temperature (see Note 5).
Rinse and wash slides three times in 1XTBST.
Rinse and wash slides one time in 1XAR6 buffer.
Transfer slides to a microwave-safe processing jar and perform microwave treatment as follows:
a) 100% power until boiling point is reached (approximately 60 s).
b) 20% power for 15 min.
c) Cool slides at room temperature for 20 min.
d) Rinse and wash slides two times in distilled water.
e) Rinse and wash slides two times in 1XTBST.
Repeat steps 2–11 until all targets have been detected. Once all targets have been detected, continue to step 13.
Prepare DAPI solution to counterstain nuclei. Add two drops of spectral DAPI per 1 mL of TBST.
Incubate slides sections DAPI for 5 min at room temperature.
Rinse and wash slides in TBST then distilled water for 2 min each.
Mount with an aqueous florescence-safe mounting medium.
Visualize slides using the Mantra Quantitative Pathology Imaging System and analyze images using InForm Visualization Software.

Notes

For optimal results, perform all incubation steps in a humidified chamber.
PerkinElmer AR9 buffer may be used for targets that require higher pH antigen retrieval.
Mouse or rabbit primary antibodies should be used with the Secondary Opal Polymer HRP Ms. + Rb. Alternate secondary antibodies should be independently validated.
Incubation times and temperatures for the primary antibodies vary and should be optimized prior to mIHC.
When deciding staining order, detect colocalizing targets with nonadjacent fluorophores for optimal results.

Prior to multiplex imaging, single-plex optimization of each fluorophore should be performed to allow for the creation of a reference spectral library.

Reference