Multiple Iterative Labeling by Antibody Neodeposition (MILAN) CURRENT STATUS: POSTED

(What’s new 5: an Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated and detail here a method based on common primary and secondary antibodies, diffusely available fluorescent image scanners and routinely processed tissue sections \(FFPE). It entails rounds of four-color indirect immunofluorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage. In excess of 50 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software.


Introduction
Staining a section with multiple antibodies satisfy two different requirements: to assess multiple analytes when the limitation is the number of sections (e.g. a single one) to classify single cells in tissue by high-dimensional methods, typically >15 markers.
Both requirements are accomplished by this method, which relies on controlled antigen retrieval conditions (once, at the beginning), antigen retention over the staining cycles, six-color immunofluorescent staining, bringing the total amount of stainings into the dozens.
It employs unconjugated primary antibodies, commercial secondary antibodies and IF microscopes and scanners.
It has thus the power to bring the two requirements mentioned above to a vast public of scientists, investigators, clinicians, etc.

2ME/SDS staining and stripping method
1 perform ARx on dewaxed sections affixed to postively charged glass slides 2 allow to cool to about 50C or lower 3 acquire the AF image for all the channels deemed necessary (optional) 3 4 perform the first IF stain in 100 mM Trehalose-containing dilution buffer 5 mount with 60% Glycerol in PBS, 0.2% N-propyl Gallate and 584 mM sucrose mounting medium containing DAPI 6 label the slide, acquire the images for all channels including DAPI and AF if not acquired before 7 unmount the slides in buffer/distilled water 8 transfer to Tris buffer 9 immerse for 30 min in pre-heated (56C) 2ME/SDS buffer with agitation 10 transfer to Tris buffer and wash extensively with TBS-Ts buffer 11 repeat from #4 with additional positive and/or negative antibodies 12 store in 50% glycerol at -20C/-80C for extended storage, before returning to #4 or proceed as  NB. An Hexane overnight step before the xylene has been recommended for complete paraffin extraction [1]: hexane is volatile and may dry the sections before entering xylene. An advantage in immunoreactivity is antigen-dependent and modest. Place in a household microwave oven (MWO), set to "high" or 850W: should boil vigorously in 8 min.
Reduce power to "low" or 300W and allow 20-30 min. of intermittent radiation to maintain boiling. This overcomes pH dependent retrieval [2] Cool to 50°C or below to allow antigen refolding before transferring to washing buffer (TBS-Ts), by checking with a kitchen thermometer. (Figure 2) Take precautions: hot fluid.
Slides can be stored in 50% glycerol-sucrose-TBS at this step (storage buffere) [3] Step Dilute all primary Ab's to 1 µg/ml (or equivalent by titration) in Antibody diluent [3] Dilute all secondary Ab's to 5 µg/ml (~1:200 -1:300). [3] have different concentration/signal curves. Alexa 488 conjugates tend not to increase signal above 5µg/ml, because of self-quenching; Rhodamine RedX and Alexa 647 do increase. BV480 conjugates tend to have an exponential increase of signal with increased concentration above 2-3 µg/ml; however, anti-isotype conjugates are much brighter than species-specific ones. If using BV480, beware of A) non-specific background increase, B) spillover of BV480 signal into Autofluorescence and FITC channels.

NB. Fluorochrome-conjugated antibodies used in double indirect IF
By using unconjugated primary antibodies in indirect immunofluorescence, the following combinations are permitted, based on species-or isotype-specific secondary antibodies and a filter combination as depicted in Fig. 5: One each of rabbit, mouse, rat and goat antibodies One rabbit Ab plus one each of the mouse IgG1, IgG2a, IgG2b or IgG3, up to one Rb + 3 mouse Abs.  Fill a standard 5 slide vertical mailer ( "Kaltek":https://www.kaltek.it/en/histology/slides/slide-mailers-2/, "MLS":https://www.mls.be/products/?lang=en&category=08&subcategory=8.1.1.9 or others) with 12 ml antibody solution. You are supposed to stain five slides simultaneously, so that the fluid can completely cover each section. This setup can be re-used for a total of approx. 10 rounds of staining (= 50 slides total) [3]; thus 12 µg of antibody is enough for one 50-slides experiment and 1 vertical mailer.

Procedure
Incubate in primary Ab overnight at room T (manual) or at +4C (mailers).
NB: overnight incubation will increase the staining efficiency [3]. Remove the disaccharide-containing fluid from the bottom of the slide with a distilled water-soaked pad, for smooth mechanical operations (may be performed just before step 10).
Affix a label containing a 2D barcode for file name reading by the instrument and with other metadata (date, experiment #, etc.) [3] Step 5: STRIPPING [3,5] Coverslip removal by soaking in coplin jar with washing buffer or distilled water (either one, OK). Transfer to Tris buffer pH 7.5, in order to remove disaccharides.
Preheat vertical containers with stripping buffer to exactly 56°C in closed, shaking water-bath. Wash at least for one hour with washing buffer with several washes in the first quarter of an hour.
Step 6: STORAGE Store at any step. If slides are not used for > 3 days store @ -20°C in storage buffer Prefer storage of unstripped slides after the last staining; stripping will get rid of autofluorescence or background formed during storage.
Step 6: N/A Troubleshooting Inadequate or failure to remove antibodies (stripping) may occur because of these causes:

Dehydration (may not be obvious on inspection, see the Ref Boi G. et al)
Expired 2-Mercaptoethanol. 2ME has a shelf-life of 36 months and a half-life of ~100 hours when diluted in a pH 6.5 buffer. Expired 2ME may still stinks, but the stripping efficiency of the 2ME/SDS mix may decrease considerably. Don't trust your nose, read the label.
Low buffer/section area ratio. In some vertical mailers, the anterior or posterior wall may bend, leaving not enough room for sufficient buffer volume to overlay the section. This may results in insufficient stripping for the sections placed first or last, particularly when the section is very large.

Anticipated Results
You should be able to sequentially stain a single FFPE section with antibodies raised in the same species, directed against close epitopes, on the very same subcellular structure, provided you space each staining by a stripping cycle.
Examples can be seen in the literature references provided.   Immunoglobulins.xlsx