TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3–7 d, depending on the type of analysis.</p>

[BEFORE_START]
<p><strong>REAGENT SETUP</strong></p>
<p> </p>
<p><strong>Definition of the target sequence </strong>Choose a region of the target mRNA that is sufficiently long so that at least 20 probes—but preferably 48—each 20 bp long, can anneal to it, with at least two bases present between neighboring probe-binding sites (which amounts to at least 1,054 bp). If possible, avoid using untranslated regions (UTRs).<strong> Δ</strong><strong>CRITICAL</strong>The presence of multiple binding sites for some of the probes in the library (other than the mRNA of interest) will lead to a high fluorescent background, which will decrease the quality of the FISH signal and may cause problems in spot detection and data analysis. If possible, exclude tandem repeats, microsatellites and other repeating sequences from the target sequence.</p>
<p> </p>
<p><strong>Design of the library of probe oligonucleotides </strong>Use the Probe Designer tool (<a href="http://www.biosearchtech.com/stellarisdesigner/" target="_blank">http://www.biosearchtech.com/stellarisdesigner/</a>) developed by Arjun Raj for this task. This tool is freely available, and no downloads are required; users only need to create their own login name and password, and then they can start using the Probe Designer immediately and free of charge. Follow stepwise instructions to upload the target sequence, to enter the information on the origin of the sequence, to specify the masking level and to create the list of oligonucleotide probes. Order the oligonucleotides on the list from Biosearch Technologies or another company of your choice. Please make sure to order sufficient quantities of the oligonucleotides in the most convenient format. In this protocol (probe-fluorophore coupling procedure, Steps 1–4), we use 10 μl of each oligonucleotide at the concentration of 100 μM (in water) per labeling reaction. In general, DNA oligonucleotides dissolved in water are stable for several years if they are stored below −20 °C. Repeated freeze-thaw cycles do not detectably affect the performance of our libraries in this method. However, please consider the manufacturer’s instructions for reconstitution and storage of the oligos.</p>
<p> </p>
<p><strong>Sodium bicarbonate, 1 M, pH 8.0 </strong>In a 50-ml Falcon tube, dissolve 4.2 g of the powder in 40 ml of distilled or deionized, nuclease-free water. Adjust the pH to 8.0 with 5 N sodium hydroxide. Adjust the volume to 50 ml. <strong>!CAUTION </strong>Sodium hydroxide is highly caustic. Handle it carefully; wear protective gloves and a lab coat. <strong>Δ</strong><strong>CRITICAL </strong>prepare this solution fresh and discard it after 2 weeks. Alternatively, divide the solution into 5-ml portions and freeze them. These aliquots can be stored at −20 °C for several months.</p>
<p> </p>
<p><strong>Sodium hydroxide, 5 N </strong>Dissolve 10 g of sodium hydroxide pellets in 50 ml of distilled or deionized water. Add the pellets in small quantities to the water, allow them to dissolve, cool the solution down, and then add the next portion of pellets. This solution can be stored indefinitely at room temperature (20–25 °C). <strong>!CAUTION </strong>Sodium hydroxide is highly caustic. Handle it carefully and wear gloves and a lab coat. Sodium hydroxide pellets absorb water from the air; weigh them quickly using glass or plastic weighing cups (not weighing paper). Dissolving sodium hydroxide is highly exothermic; keep the tube on ice. Do not pour water onto the pellets, as it will boil and splash out of the tube.</p>
<p> </p>
<p><strong>Sodium bicarbonate, 0.1 M, pH 8.0 </strong>Combine 5 ml of the 1 M sodium bicarbonate and 45 ml of distilled or deionized nuclease-free water. <strong>Δ</strong><strong>CRITICAL </strong>Freshly prepare this solution each time or make sure to use it within 1 week of preparation.</p>
<p> </p>
<p><strong>HPLC buffer A </strong>Mix 100 ml of 1 M triethylammonium acetate with 900 ml of deionized water. Freshly prepare the buffer every week and store it at room temperature. <strong>!CAUTION </strong>Triethylammonium acetate is volatile; handle it in a fume hood while wearing protective gloves.</p>
<p> </p>
<p><strong>HPLC buffer B </strong>HPLC buffer B is undiluted acetonitrile. <strong>!CAUTION </strong>This is an organic solvent; handle it in a fume hood while wearing protective gloves.</p>
<p> </p>
<p><strong>DAPI, 20 mg ml </strong><strong>−1 </strong>Add 500 μl of deionized, nuclease-free water to 10 mg of DAPI powder. Divide the solution into 20- to 50-μl portions and store them at −20 °C for up to several years.</p>
<p> </p>
<p><strong>DAPI, 100 </strong>μ<strong>g ml </strong><strong>− 1 </strong>Add 990 μl of deionized, nuclease-free water to 10 μl of 20 mg ml −1 DAPI. Store the solution at 4 °C and use it within a month.</p>
<p> </p>
<p><strong>Formaldehyde, 3.7% (vol/vol) </strong>For a 50-ml solution, combine 40 ml of distilled or deionized, nuclease-free water, 5 ml of 37% formaldehyde (wt/vol) and 5 ml of 10× PBS. Freshly prepare the solution just before use; </p>
<p>alternatively, prepare it in advance, divide it into 25- to 50-ml portions and freeze the portions. Store formaldehyde at −20 °C for up to several months and thaw it just before use. <strong>!CAUTION </strong>Formaldehyde is highly toxic and carcinogenic. Handle it in a fume hood while wearing a lab coat and protective gloves.</p>
<p> </p>
<p><strong>Paraformaldehyde, 4% (vol/vol) </strong>Combine 10 ml of 16% (wt/vol) paraformaldehyde, 4 ml of 10× PBS and 26 ml of deionized, nuclease-free water. Freshly prepare the solution just before use or prepare it in advance, divide it into 25- to 50-ml portions and store the portions at −20 °C for up to several months. Thaw the solution in a 37 °C water bath just before use. <strong>!CAUTION </strong>Paraformaldehyde is highly toxic and carcinogenic. Handle it in a fume hood while wearing a lab coat and protective gloves.</p>
<p> </p>
<p><strong>Cryoprotecting solution </strong>Combine 5 ml of 10× PBS, 5 ml of 37% (vol/vol) formaldehyde and 15 g of sucrose. Add distilled or deionized, nuclease-free water to a final volume of 50 ml. Incubate the solution with gentle agitation at room temperature to allow the sucrose crystals to dissolve. Freshly prepare this solution just before use or prepare it in advance, divide it into 25- to 50-ml portions and store the portions at −20 °C for up to several months. Thaw the solution in a 37 °C water bath just before use. <strong>!CAUTION </strong>Formaldehyde is highly toxic and carcinogenic. Handle it in a fume hood while wearing a lab coat and protective gloves.</p>
<p> </p>
<p><strong>RBS-35, 2% (vol/vol) </strong>Combine 20 ml of RBS and 980 ml of distilled or deionized water. Store the solution at room temperature for up to several months.</p>
<p> </p>
<p><strong>Ethanol, 70% (vol/vol) </strong>Combine 700 ml of absolute ethanol and 300 ml of distilled or deionized nuclease-free water. Store it indefinitely in a tightly closed container at room temperature.</p>
<p> </p>
<p><strong>Poly-</strong><strong>l</strong><strong>-lysine </strong>Combine 20 ml of 0.1% (wt/vol) poly-l-lysine solution and 180 ml of deionized or distilled, nuclease-free water. Freshly prepare the solution before use and discard the remaining solution after use.</p>
<p> </p>
<p><strong>Ribonucleoside vanadyl complex </strong>Incubate the purchased solution in a 65 °C water bath for 10 min, divide it into 100-μl aliquots and store them at −20 °C for up to several months.</p>
<p> </p>
<p><strong>Glucose oxidase, 100× stock </strong>Dissolve the enzyme in 50 mM sodium acetate (pH 5.0), to a concentration of 3.7 mg ml −1. Divide the solution into 50- to 100-μl aliquots and store them at −20 °C for up to several months.</p>
<p> </p>
<p><strong>Trolox, 200 mM (100× stock) </strong>Dissolve 5 mg of the Trolox powder in 1 ml of pure ethanol. Divide the solution into 50- to 100-μl portions and store them at −20 °C for up to several months.</p>
<p> </p>
<p><strong>Glucose 10% (wt/vol) </strong>Dissolve 5 g of glucose in 50 ml of deionized, nuclease-free water. Sterilize the solution by passing it through a 0.2-μm syringe filter and store it at 4 °C for up to a few weeks.</p>
<p> </p>
<p><strong>GLOX buffer </strong>Combine 100 μl of Tris (1 M, pH 8.0), 1 ml of 20× SSC, 400 μl of 10% (wt/vol) glucose and 8.5 ml of deionized, nuclease-free water. This solution needs to be prepared freshly at least every week. For best results, prepare the solution just before use.</p>
<p> </p>
<p><strong>GLOX buffer with Alexa Fluor 488 phalloidin </strong>Reconstitute Alexa Fluor 488 phalloidin as described in the manufacturer’s manual. Briefly, add 1.5 ml of methanol to 300 units of Alexa Fluor 488 phalloidin and dissolve it by passing it several times through a pipette tip. Add 4 μl of the reconstituted Alexa Fluor 488 phalloidin to 2 ml of GLOX buffer. Freshly prepare this solution before use.</p>
<p> </p>
<p><strong>Imaging buffer </strong>Add 1 μl of glucose oxidase (stock 3.7 mg ml − 1), 1 μl of catalase (vortex catalase suspension just before pipetting, do not spin) and 1 μl of Trolox (200 mM stock) to 100 μl of GLOX buffer. Use this solution immediately, although it may be stored on ice or at 4 °C for up to 8 h.</p>
<p> </p>
<p><strong>Formamide </strong>Before opening the formamide bottle, bring it to room temperature. Divide the solution into 10- to 50-ml portions and store them in the dark at 4 °C for up to several months. <strong>!CAUTION </strong>Formamide is toxic and teratogenic. Handle it in a fume hood while wearing protective gloves and a lab coat.</p>
<p> </p>
<p><strong>Wash buffer, 10% (vol/vol) formamide </strong>Combine 5 ml of 20× SSC, 5 ml of formamide and 45 ml of deionized, nuclease-free water. Freshly prepare the solution before use. <strong>!CAUTION </strong>Formamide is toxic and teratogenic. Handle it in a fume hood while wearing protective gloves and a lab coat.</p>
<p> </p>
<p><strong>Wash buffer with DAPI </strong>Prepare the wash buffer, 10% (vol/vol) formamide, as described above. Add 50 μl of DAPI (100 μg ml − 1 stock) to 10 ml of wash buffer (1:200 dilution). Use this solution immediately. <strong>!CAUTION </strong>Formamide is toxic and teratogenic. Handle it in a fume hood while wearing protective gloves and a lab coat.</p>

[GUIDELINES]
<p style="text-align: justify;">The protocol workflow timing is as follows:</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Steps 1–5</strong>, coupling FISH probe libraries to fluorophores: 2–4 h</p>
<p style="text-align: justify;"><strong>Steps 6–10</strong>, probe precipitation, washing, and purification: 1–2 d</p>
<p style="text-align: justify;"><strong>Step 11</strong>, probe reconstitution and storage: 5–10 min</p>
<p style="text-align: justify;"><strong>Steps 12–16</strong>, tissue dissection, fixing and cryoprotection: 4 h to overnight</p>
<p style="text-align: justify;"><strong>Steps 17–20</strong>, preparation of frozen blocks: 30–40 min</p>
<p style="text-align: justify;"><strong>Steps 21–26</strong>, cryosectioning, fixation and permeabilization of the sections: 5 h 30 min to overnight</p>
<p style="text-align: justify;"><strong>Steps 27–29</strong>, proteinase K digestion (optional): 20 min</p>
<p style="text-align: justify;"><strong>Steps 30–32</strong>, hybridization: overnight</p>
<p style="text-align: justify;"><strong>Steps 33–36</strong>, washing and mounting: 1 h 40 min</p>
<p style="text-align: justify;"><strong>Steps 37–42</strong>, microscope setup and data collection: 4–6 h</p>
<p style="text-align: justify;"> </p>
<p> </p>
<p><strong>REQUIRED EQUIPMENT</strong></p>
<p> </p>
<ul>
<li>Microcentrifuge tubes, 1.8 mg</li>
<li>Six-well plates (BD Biosciences, cat. no. 351146)</li>
<li>Petri dishes (BD Biosciences, cat. no. 351029)</li>
<li>Parafilm (Sigma-Aldrich, cat. no. P7793-1EA)</li>
<li>Sharp-tip forceps (ROBOZ, cat. no. RS-5095)</li>
<li>Filter paper (Schleicher &amp; Schuell, cat. no. 10310992)</li>
<li>Speed vacuum concentrator (Eppendorf Concentrator Plus)</li>
<li>Tabletop centrifuge (Eppendorf, cat. no. 5424R)</li>
<li>Vortex Genie (Scientific Industries)</li>
<li>NanoDrop 2000 spectrophotometer (Thermo Scientific)</li>
<li>Falcon tubes, 15 mg (BD Biosciences, cat. no. 352196)</li>
<li>Falcon tubes, 50 mg (BD Biosciences, cat. no. 352070)</li>
<li>Tube rotator (Stuart, cat. no. SB2)</li>
<li>Nutating mixer (VWR)</li>
<li>Centrifuge (Eppendorf, cat. no. 5810R)</li>
<li>RNase-free, low-retention pipette tips (GeneMate, cat. no. P-1237)</li>
<li>Sonicator (VWR, Ultrasonic Cleaner)</li>
<li>Desiccator</li>
<li>Plastic molds for tissue embedding, cat. no. 6254-10 (10 × 10 × 5 mm); cat. no. 6254-15 (15 × 15 × 5 mm); cat. no. 6254-25 (25 × 20 × 5 mm) (frozen blocks; Tissue Tek Cryomold, Electron Microscopy Sciences)</li>
<li>Cryostat (Cryostar NX70, Thermo Scientific)</li>
<li>HPLC (Gilson GX271 liquid handler, with C18 Targa column and UV-visible 155 spectrometer, Higgins Analytical)</li>
<li>Pre-column frit (Chrom Tech, cat. no. A-332X)</li>
<li>Hybridization oven (UVP, HB-1000 hybridizer)</li>
<li>Microscope glass slides (Thermo Scientific, Gold Seal, cat. no. 3031-002)</li>
<li>Microscope cover glass, 22 × 22 mm, no. 1 (Thermo Scientific, cat. no. D10143263NR1)</li>
<li>Sterile syringe filters, 0.2 μm (Millipore, cat. no. 6900-2502)</li>
<li>Cover glass staining rack (Electron Microscopy Sciences, cat. no. 72241-01)</li>
<li>Cover glass wash jar (Electron Microscopy Sciences, cat. no. 70312-23)</li>
<li>Inverted fluorescence microscope (Nikon, Eclipse-Ti)</li>
<li>Numerical aperture (NA) 1.4 objective, ×100 (Nikon)</li>
<li>Immersion oil</li>
<li>Metal halide lamp (Prior Lumen 220)</li>
<li>High sensitivity CCD camera (Photometrics Pixis 1024B)</li>
<li>Motorized optical shutter (Sutter SMARTSHUTTER, Sutter Instruments)</li>
<li>Motorized stage controller (TI-S-E motorized stage)</li>
<li>Filter cube for Cy5.5 (Chroma Technology, cat. no. 41023)</li>
<li>Custom filter cube for Alexa Fluor 594: Omega Optical (exciter: cat. no. 2017901-590DF10, dichroic filter: cat. no. XF2014-610DRLP and emitter: cat. no. XF3028-630DF30)</li>
<li>Filter cube for TMR (Omega Optical, cat. no. XF204)</li>
<li>Computer of minimum 4 GB RAM</li>
<li>Microscope managing and imaging software (Metamorph, Molecular Devices)</li>
<li>MATLAB (The MathWorks)</li>
<li>KNF Teflon pump</li>
<li>Vacuum pump</li>
<li>Styrofoam boxes with lids</li>
<li>ImageM and other MATLAB codes developed in our lab are available upon request (requests can be addressed to S.I. (shalev.itzkovitz@weizmann.ac.il), J.P.J. (j.junker@hubrecht.eu) and A.v.O. (a.oudenaarden@hubrecht.eu))</li>
</ul>
<p> </p>
<p style="text-align: justify;"><strong>Applications of the method</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">This protocol enables researchers to determine the absolute numbers of mRNA molecules in single cells within a tissue. The absolute numbers obtained by our technique are in a close correspondence to those measured by different means, for instance, via quantitative reverse-transcription PCR (qRT-PCR)1,2. As few as one mRNA molecule per cell can be detected. The maximum number of individual molecules that can be resolved microscopically is of the order of several thousand. This upper limit is dictated by the ratio between the cell volume and the typical voxel taken by a diffraction-limited mRNA dot (~0.2 × 0.2 × 0.3 μm).</p>
<p style="text-align: justify;">Three different mRNA species can be simultaneously visualized (using probe libraries differentially labeled with Cy5, Alexa Fluor 594 and tetramethylrhodamine (TMR)) and quantified in parallel by implementing the present protocol; this upper limit is determined by the number of different fluorophores that can be spectrally resolved. Simultaneous staining with a DNA-binding dye and an antibody against cell-cell adhesion proteins enables the visualization of cell nuclei and borders,</p>
<p style="text-align: justify;">which is a requirement when the aims are to assign mRNA spots to cells within intact tissue.</p>
<p style="text-align: justify;">As an example of quantitative and spatial analysis of gene expression, here we use the crypt of Lieberkühn, a functional unit of the intestinal epithelium, as a target tissue. First, the numbers of diffraction-limited spots are automatically detected using algorithms previously developed in our laboratory2,3. Next, cells are manually segmented on the basis of DAPI nuclear staining and E-cadherin lateral membrane staining, and spots are then assigned to individual cells. The resulting single-cell gene-expression profiles can be presented as a spatial profile (<strong>Fig. 1</strong>), which we termed ‘cryptogram’, from ‘crypt’ and ‘histogram’. This approach enables the experimenter to assess the precise spatial location of the expression of genes of interest in multiple crypts in the tissue of interest3 (<strong>Fig. 1h</strong>).</p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rphfw26.png" width="584" height="480" data-src="https://s3.amazonaws.com/pr-journal/rphfw26.png" data-ofn="Screen Shot 2017-12-21 at 1.56.14 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">In addition to the intestine3–6, the three-color smFISH protocol can be applied to a variety of tissues, including mammary glands7, kidney8, stomach, lymph nodes, mouse embryos at different gestational stages, human breast tumors9 and possibly many more. Although the quality of FISH signal is consistently high in the different tissues tested, autofluorescence and/or nonspecific probe binding may emerge in some tissues, and it may need to be addressed if it interferes with spot detection and/or data analysis. Examples of tissues that we have found to be particularly high in autofluorescence are skin (especially hair shafts and cornified layers of epidermis; A.L., unpublished observation), stomach (parietal cells contain highly autofluorescent cytoplasmic vesicles; A.L., unpublished observation) and small intestine (Paneth cells with highly autofluorescent cytoplasmic granules3). smFISH can also be combined with other techniques, such as EdU staining4, immunofluorescence4 and DNA FISH9 for probing single cells in intact tissues.</p>
<p style="text-align: justify;">Given that hybridization conditions for different probe sets are similar, this method has great potential for the simultaneous detection of several mRNA species. As mentioned, the number of mRNA species to be detected simultaneously is limited by the availability of fluorophores that can be spectrally resolved using current technology. We routinely detect three different transcripts at the same time, using Cy5, Alexa Fluor 594 and TMR fluorophores. Other fluorophores emitting light at different wavelengths can be used as well, thus increasing the number of mRNAs that can be simultaneously detected10. Furthermore, combinatorial labeling, in which different probe subsets are coupled to different fluorophores, can further increase the number of different color combinations that can be computationally resolved10,11. This approach may enable truly multiplexed single-molecule mRNA imaging, which would greatly facilitate large-scale gene expression profiling in single cells, in the context of tissues.</p>
<p style="text-align: justify;">Cell border detection (segmentation) may become a challenge in tissues for which no good antibody against cell membrane– associated proteins is available. In these cases, cell borders may be visualized by other means, such as by using fluorescently labeled phalloidin or GFP-fusion membrane-associated proteins. An example of the latter is the use of tissues from a knock-in mouse expressing E-cadherin fused to cyan fluorescent protein (CFP)12. Alternatively, when cell border labeling is impossible, too laborious or not crucial, tissue surface can be divided into arbitrary (virtual) sectors (‘cell proxies’) and mRNA spots can be assigned to these proxies instead of cells. After the segmentation step, spatial analysis of mRNA distributions can be performed according to the topological features of the tissue.</p>
<p style="text-align: justify;">In addition to tissue sections, our protocol can be applied to whole-mount organs or embryos with no substantial modifications. The only challenging part of such an application is imaging depth, as the smFISH signal quality from parts lying deeper than 10 μm below the objective will decrease substantially. This problem can be alleviated by using confocal microscopy. The best results so far are obtained on a spinning-disk microscope (quantifiable signal from tissue layers 30–50 μm deep tissue).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rpifw26.png" width="579" height="333" data-src="https://s3.amazonaws.com/pr-journal/rpifw26.png" data-ofn="Screen Shot 2017-12-21 at 1.59.36 PM.png" /></p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rpjfw26.png" width="580" height="222" data-src="https://s3.amazonaws.com/pr-journal/rpjfw26.png" data-ofn="Screen Shot 2017-12-21 at 1.59.52 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Experimental design</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Oligonucleotide libraries are designed in agreement with the set of requirements established previously2, and then purchased. By the term ‘library’, we mean a set of oligonucleotides, each of which hybridizes to the same mRNA molecule. The ‘collective’ binding of singly labeled oligonucleotides to the target mRNA molecule results in the appearance of a single bright fluorescent spot. A typical library consists of 48 oligonucleotides, each 20 bp long. The number of oligonucleotides in a library should exceed 20 (ref. 2), and it often is as large as 96.</p>
<p style="text-align: justify;">The minimum size of a library depends on the length of the target mRNA. This target molecule should be sufficiently long to accommodate at least 20 oligonucleotides, plus a two-base space between neighboring oligonucleotides. Other important parameters are the G-C content of the library, which should be as close to 45% as possible, and the uniqueness of the target sequence (to minimize binding of the probes to anything other than the target mRNA). Conveniently, the ‘Probe Designer’ tool (freely available at <a href="http://www.biosearchtech.com/stellarisdesigner/" target="_blank">http://www.biosearchtech.com/stellarisdesigner/</a>) will guide a user through probe design, taking all the important parameters into consideration.</p>
<p style="text-align: justify;">The 3′-end of each library oligonucleotide is coupled to a single fluorophore (Steps 1–5), and the resulting labeled oligonucleotide is purified (Steps 6–10). Alternatively, the sets of fluorophore-coupled and purified oligonucleotides can be purchased from Biosearch Technologies under the brand name Stellaris, or from another company of choice.</p>
<p style="text-align: justify;">The tissue is dissected right after euthanizing the animal, and it is immediately fixed (Steps 12–16). After a cryoprotection step (Step 16), frozen blocks are prepared (Steps 17–20). Cryosections of 5–10-μm thickness are mounted onto cleaned and poly-l-lysine–coated glass coverslips (Steps 21–26). The attached sections are fixed again, rinsed and permeabilized (Steps 24–26), and then rehydrated and hybridized with the probes and antibody overnight (Steps 30–32). An antibody can be used to stain the cell borders, so as to visualize tissue structure. However, if hybridization conditions are not optimal for staining with the necessary antibody, alternative counterstaining methods should be considered, such as staining with fluorescently labeled phalloidin (Step 35). After hybridization, sections on coverslips are washed, counterstained with nuclear dye and equilibrated with the imaging buffer. If phalloidin staining is chosen to visualize the cell border, this staining is performed at this point, and sections are subsequently rinsed and mounted (Steps 33–36).</p>
<p style="text-align: justify;">Images are acquired using an inverted epifluorescence microscope equipped with an automated stage, a cooled back-illumined charge-coupled device (CCD) camera and a ×100 oil-immersion objective with a numerical aperture of 1.4 (Steps 37–39). At each position, a 5–10-μm-thick <em>z</em>-stack of images with 0.2–0.4 μm of distance between individual planes is acquired in order to collect the information from the whole thickness of the section.</p>
<p style="text-align: justify;">The data are analyzed using MATLAB software (Steps 40–42). This software enables the automatic detection of the mRNA molecule spots, assists in manually segmenting cell borders and assigns the spots to individual cells, labeled according to position within the tissue relative to a reference (for instance, the bottom of an intestinal crypt). All automated and manual steps are integrated into ImageM, a straightforward and user-friendly GUI developed in our lab (<strong>Box 1 </strong>and <strong>Figs. 2 </strong>and <strong>3</strong>).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rpkfw26.png" width="287" height="171" data-src="https://s3.amazonaws.com/pr-journal/rpkfw26.png" data-ofn="Screen Shot 2017-12-21 at 2.03.04 PM.png" /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rpmfw26.png" width="275" height="110" data-src="https://s3.amazonaws.com/pr-journal/rpmfw26.png" data-ofn="Screen Shot 2017-12-21 at 2.03.17 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rppfw26.png" width="346" height="202" data-src="https://s3.amazonaws.com/pr-journal/rpnfw26.png" data-ofn="Screen Shot 2017-12-21 at 2.05.55 PM.png" /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rpqfw26.png" width="212" height="121" data-src="https://s3.amazonaws.com/pr-journal/rpqfw26.png" data-ofn="Screen Shot 2017-12-21 at 2.06.12 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Key analysis of the gene expression signatures created can include spatial mapping of gene expression, single-cell gene expression profiles and single-cell correlations of pairs of genes3. In intestinal crypts, the position of cells above the crypt apex defines a natural one-dimensional axis. For each gene, a spatial gene expression profile can be created. Pooling profiles from different analyzed crypts enables one to obtain the average gene expression profile and its variance, which we term a cryptogram (<strong>Fig. 1g,h</strong>). Such an analysis is particularly suited to genes broadly expressed in a consistent manner between crypts. Separate analysis can be applied to other genes that show a more variable expression pattern in terms of the position of the expressing cells along the crypt axis.</p>
<p style="text-align: justify;">In the mouse intestine, some genes are highly specific to particular differentiated cell types, which can appear in variable positions in different crypts. Examples include markers for Goblet cells (<em>Clca3</em>) and Tuft cells (<em>Dclk1</em>, also known as <em>Dcamkl1</em>)3. An additional analysis, which can be performed using smFISH on tissue sections, is the characterization of single-cell gene expression signatures for such rarer cell types. The numbers of transcripts of other genes in such cells, defined by a marker of interest, can be compared to cells, which are directly above or below the cell of interest. This analysis stringently tests the upregulation or downregulation of particular genes in the cell type of interest, while controlling the spatial pattern of gene expression of the tested genes3. In addition, the Pearson or Spearman correlation coefficients of </p>
<p style="text-align: justify;">the single-cell gene expression, correlated transcription patterns of pairs of genes may indicate the presence of common regulatory modules for these genes.</p>
<p style="text-align: justify;">More advanced and sophisticated analyses can be performed on the resulting data sets. MATLAB codes are available upon request (see MATERIALS).</p>
<p style="text-align: justify;">Finally, please note that although positive and/or negative controls for the expression of specific mRNAs are not covered in the procedure detailed below their use is likely to greatly improve the reliability of the protocol’s results. A more in-depth discussion on this subject is reported in the ANTICIPATED RESULTS (Step 42).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>TROUBLESHOOTING </strong></p>
<p style="text-align: justify;"> </p>
<table style="height: 235px; width: 649px;">
<tbody>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;"><strong>Step </strong></td>
<td style="width: 159px; height: 20px; text-align: justify;"><strong>Problem </strong></td>
<td style="width: 211px; height: 20px; text-align: justify;"><strong>Possible reason </strong></td>
<td style="width: 215px; height: 20px; text-align: justify;"><strong>Solution </strong></td>
</tr>
<tr style="height: 164px;">
<td style="width: 48px; height: 164px; text-align: justify;">25-35</td>
<td style="width: 159px; height: 164px; text-align: left;">Tissue sections detach from the glass</td>
<td style="width: 211px; height: 164px; text-align: left;">Insufficient cleaning and/or coating of the coverslips</td>
<td style="width: 215px; height: 164px; text-align: left;">Increase the length of coverslip sonication in detergent and ethanol to 20 min or add a second sonication step in ethanol. Make sure the coated coverslips are used immediately, or that they are stored in the desiccator only</td>
</tr>
<tr style="height: 36px;">
<td style="width: 48px; height: 36px; text-align: left;"> </td>
<td style="width: 159px; height: 36px; text-align: left;"> </td>
<td style="width: 211px; height: 36px; text-align: left;">Insufficient drying at Step 23</td>
<td style="width: 215px; height: 36px; text-align: left;">Increase drying time by 1–2 min. Try not to exceed 8 min</td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;">37</td>
<td style="width: 159px; height: 20px; text-align: left;">Tissue integrity is poorly preserved, with frequent rips and holes throughout the samples</td>
<td style="width: 211px; height: 20px; text-align: left;">Tissue is cryodamaged</td>
<td style="width: 215px; height: 20px; text-align: left;">Cryoprotection step is necessary (Step 16)<br /><br /></td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;"> </td>
<td style="width: 159px; height: 20px; text-align: left;">Tissue is twisted and/or ripped</td>
<td style="width: 211px; height: 20px; text-align: left;">Sample was damaged during mounting (Step 36)</td>
<td style="width: 215px; height: 20px; text-align: left;">Do not let coverslip move during mounting</td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;">38,39</td>
<td style="width: 159px; height: 20px; text-align: left;">Insufficient signal intensity (low signal-to-background ratio)</td>
<td style="width: 211px; height: 20px; text-align: left;">Nonspecific probe binding owing to a suboptimal formamide concentration, leading to a high background fluorescence</td>
<td style="width: 215px; height: 20px; text-align: left;">Increase formamide concentration up to 25% (vol/vol) in wash and hybridization buffers (the required formamide concentration can be probe dependent)</td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;"> </td>
<td style="width: 159px; height: 20px; text-align: left;"> </td>
<td style="width: 211px; height: 20px; text-align: left;">Insufficient number of binding probes due to the association of target mRNA with proteins</td>
<td style="width: 215px; height: 20px; text-align: left;">Subject tissue sections to proteinase K digestion (Steps 27–29). Increase proteinase K concentration, the duration of digestion and the incubation temperature, if needed, being careful not to affect tissue integrity</td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;"> </td>
<td style="width: 159px; height: 20px; text-align: left;"> </td>
<td style="width: 211px; height: 20px; text-align: left;">Insufficient number of binding probes due to a small oligonucleotide library size</td>
<td style="width: 215px; height: 20px; text-align: left;">If possible, increase the size of the library, such as by adding UTRs to the target sequence</td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;"> </td>
<td style="width: 159px; height: 20px; text-align: left;"> </td>
<td style="width: 211px; height: 20px; text-align: left;">High autofluorescence</td>
<td style="width: 215px; height: 20px; text-align: left;">
<p> Use red dyes, such as Cy5 instead of AlexaFluor594 and TMR. Autofluorescence is generally weaker at longer wavelengths, improving signal-to-noise </p>
</td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;"> </td>
<td style="width: 159px; height: 20px; text-align: left;">Photobleaching (signal quickly fades during acquisition)</td>
<td style="width: 211px; height: 20px; text-align: left;">Antiphotobleaching medium is not working properly</td>
<td style="width: 215px; height: 20px; text-align: left;">Prepare fresh GLOX buffer. Make sure glucose oxidase and catalase are fresh, properly reconstituted and stored. Activity of these enzymes is crucial to buffer performance. Use a lower intensity of excitation light, in particular when using laser illumination in (spinning-disk) confocal microscopy</td>
</tr>
<tr style="height: 20px;">
<td style="width: 48px; height: 20px; text-align: justify;"> </td>
<td style="width: 159px; height: 20px; text-align: left;">Intensity of fluorescent dots decays toward the edges of the image, rendering accurate quantification difficult</td>
<td style="width: 211px; height: 20px; text-align: left;">Uneven illumination of the sample (vignetting)</td>
<td style="width: 215px; height: 20px; text-align: left;">Optimize alignment of the illumination, apply digital shading correction, or exclude peripheral areas from analysis</td>
</tr>
</tbody>
</table>
<p> </p>
<p style="text-align: justify;"> </p>
<p><strong>For Comparison with other methods, </strong><strong>Limitations, References, and more information please see the attached full manuscript.</strong></p>

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