Protocol to detect RNAs from tissue sections in mice using Y-branched probe insitu hybridization

Summary Here, we describe a fluorescent in situ hybridization protocol named Yn-situ, standing for Y-branched probe in situ hybridization, to detect RNAs from mice tissue sections. We provide steps for the synthesis and quantification of preamplifier probe using nickase. We also detail the preparation of tissue section, probe hybridization, signal development using hybridization chain reaction (HCR), and quantification of the signals. This approach avoids the use of proprietary in situ hybridization techniques, therefore reducing costs. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022).

Note: A simple way to calculate the volume of IDTE buffer (in mL) to be added to the tube is to multiply the amount of oligos (in nmole) by 5. For example, for a tube containing 10 nmole oligos, dissolve it in 50 mL of IDTE buffer. 8. Mix equal volume of all odd probes into one tube and dilute the mixture using IDTE to make each probe 2 mM.
Note: Preparation will depend on the number of probes to be mixed. For a 10 nmole scale synthesis, if 5 pairs of primary probes are used, add 50 mL of each 200 mM odd probe into a 15 mL conical tube. Add 4.75 mL IDTE buffer to a final volume of 5 mL. A quick look up table is provided below step 9.
9. Mix equal volume of all even probes into one tube and dilute the mixture using IDTE to make each probe 2 mM.

Timing: 2 days
This process is for the synthesis of preamplifier probes used in Yn-situ. An illustration of the procedure is provided in Figure 2.
Note: This protocol uses QIAGEN Maxiprep kit for plasmid purification. The following reagents refer to the components in the QIAGEN Maxiprep kit.
CRITICAL: P004 confers kanamycin resistance. It is critical to use the correct antibiotics in the agar plate and LB media during the plasmid preparation.
b. Select a few single colonies to perform miniprep and make glycerol stocks of the selected clones. c. Verify the number of B1 initiator by sequencing.
CRITICAL: P004 contains 20 repeats of the B1 initiator sequence and can potentially result in recombination in the bacteria. It is critical to verify that the sequence is intact.
d. Select a clone with the correct number of B1 initiators. e. Inoculate 20 mL of glycerol stock in 300 mL of L.B. medium containing 50 mg/mL of kanamycin overnight (12-18 h) at 37 C, 220 rpm.  Note: For convenience, NEB stable cells containing P004 can be grown at 37 C to shorten the growth time and increase maxiprep yield. Although it is recommended to grow NEB stable cells at 30 C, we do not find high frequency of recombination among the B1 initiator repeats when the cells are growth at 37 C.
f. Pellet the cells at 12,000 3 g, 4 C, for 5 min. g. Resuspend the pellet in 10 mL buffer P1. h. Add 10 mL of buffer P2. i. Gently mix the solution by slowly inverting the tube. j. Lyse the cells on ice for 5 min.
Note: The preamplifier is eventually purified by gel extraction. The residual genomic DNA and RNA are removed during the gel excision step. However, significant amount of genomic DNA does affect the quantification of plasmid, which may affect estimation of plasmid quantity for the nicking step. Therefore, we recommend following the specified lysis timing. CRITICAL: The digestion should be performed at 50 C, not 37 C. The reaction should not be performed overnight (12-18 h) to avoid star activity.
Note: This step can be performed in a thermocycler.
Note: The DNA is purified using Zymo DNA clean and concentrator kit. The following reagents refer to the components in the kit. f. Run the gel at 5 V/cm (100 V for a standard 20 cm long gel tank) for 1 h. See troubleshooting: problems 2, 3, and 4.
Note: it is not necessary to run a denaturing gel. We find the ssDNA separates as long as they are denatured before loading into the gel.
Note: The DNA is purified using Zymo gel extraction kit. The following reagents refer to the components in the kit. a. Make a 1:10 dilution of the 1 kb plus ladder by adding 1 mL of ladder to 9 mL of ddH2O. b. Make a 1:100 dilution of the ladder by adding 1 mL of the 1:10 dilution ladder into 9 mL of ddH2O. c. Make a 1:10 dilution of preamplifier by adding 1 mL of purified preamplifier to 9 mL of H 2 O. d. In an 8-tube PCR strip, mix according to the table below by adding the specified amount of sample and DMSO to the tubes.
Note: The concentration of the 850 bp band is 30 ng/mL in undiluted molecular marker mix. Following this protocol, 0.3 ng, 0.6 ng, 1.5 ng, 3 ng, 6 ng, and 15 ng of the 850 bp band will be present in the gel. To measure the concentration of preamplifier accurately, it is optimal that 1-10 ng of preamplifier is loaded to the gel. Without considering loss, the theoretical yield of preamplifier from 40 mg plasmids is around 80 ng/mL.

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Note: Supplementary file Data S2 performs linear regression in excel based on the 850 bp bands from the ladder lanes and automatically calculate the concentration of the preamplifier. It is designed for non-expert to use. Alternative methods can also be used.
t. The concentration of the preamplifier band will appear. u. Dilute preamplifier to 20 ng/mL according to the measured concentration of the stock solution.
CRITICAL: The concentration of the preamplifier is critical to Yn-situ and must be measured accurately. We recommend performing gel quantification as it detects ssDNA with high sensitivity. Alternatively, a Qubit fluorometer with a ssDNA quantification kit can be used. However, a typical yield of preamplifier under the current protocol is between 2-10 ng/mL which is at the lower end of the Qubit ssDNA detection range. Quantification by Nanodrop spectrometer is frequently not accurate because of the residual agarose melting buffer.

Alternatives:
We have compared several dyes including SYBR safe, Midori green, ethidium bromide, and GelRed. We find the staining sensitivity of SYBR safe and Midori green is significantly lower than GelRed. We also find ethidium bromide and Midori green migrate toward cathode, causing the low molecular weight bands to be lower in brightness. We do not recommend using them as alternatives.

MATERIALS AND EQUIPMENT
Alternatives: This protocol uses Boekel Shaking Hybridizing Incubator for hybridization steps.
We have also acquired good results using Stratagene PersonalHyb incubator and ACD HybEZ Hybridization System.
CRITICAL: This buffer needs to be prepared fresh for each experiment.
CRITICAL: 1-methlyimidazole is a toxic chemical and should be handled and disposed of safely.
Note: The solution can be stored at À20 C for 1 year. It may be warmed up to 37 C to dissolve ETT.
CRITICAL: ETT is a toxic and self-reactive chemical and should be handled and disposed of safely. CRITICAL: This buffer needs to be prepared fresh for each experiment.
CRITICAL: EDC is a toxic chemical and should be handled and disposed of safely.
Note: This solution can be stored at room temperature (18 C-25 C) for 1 year.
Note: This solution can be stored at À20 C for 1 year.
Note: This solution can be stored at À20 C for 1 year.
CRITICAL: Formamide and the buffer containing formamide are toxic and should be handled and disposed of safely.

STEP-BY-STEP METHOD DETAILS Tissue preparation
Timing: 1 h Tissue is collected from the animal, snap frozen in O.C.T. using liquid nitrogen vapor, and stored at À80 C.
1. Sacrifice the animal by cervical dislocation. 7. Store the block at À80 C until use. The frozen sample can be stored up to a year.
Note: This step is optional. The tissue block can be processed directly to the next step if the user chooses not to store the sample.

Timing: 4 h
Frozen tissue is sectioned into 10 mm sections and mounted on slides. The tissue sections are fixed for long term storage.
8. Section the tissue at 10 mm using a cryostat at À25 C.
Note: The number of sections that can fit onto a single slide depends on the size of the tissue and the skill of the experimenter. For example, for sections 0.5 cm in length and width, 4-8 sections may be processed on one slide. 9. Dry slides at 100 C for 2 min and proceed directly to fixation.
Note: To avoid drying of the solution in the following steps, the slides are processed in a humidifying chamber, a small plastic box with added water or wet Kimwipe at the bottom, as indicated in the Figure 3.
10. Add water into the humidifying chamber. 11. Place the slides into the humid chamber. The slides should be elevated above the water at the bottom and leveled. 12. Fix slides in 4% PFA for 1 h at room temperature (18 C-25 C). 13. Wash slides 33 with PBS, 5 min each.  14. Wash slides with freshly prepared 1-methylimidazole buffer for 5 min. 15. Fix slides in EDC fixative for 1 h. 16. Wash slides with PBS for 5 min.
CRITICAL: PFA and EDC are toxic and should be handled and disposed of safely.
Optional: Permeabilize slides in 100% methanol at room temperature (18 C-25 C) for 1 h. For eye tissue, this step improves probe penetration.

Remove PBS.
Pause point: The slides can be store at À80 C after removing PBS. If methanol permeabilization is used, the tissue sections can be stored directly in methanol.

Timing: 1-2 days
This step is the core procedure for detection of RNA using Yn-situ. The target RNA first hybridizes with targeting probes. The preamplifier then hybridizes with targeting probe pairs that bind close to each other. Fluorescent signals are developed by HCR reaction in situ.

Air dry the slides and draw a well around the tissue with ImmEdge hydrophobic barrier PAP pen.
Note: The hydrophobic barrier should be complete dry before the next step to ensure its proper function (Figure 4).
19. Digest the tissue with 10 mg/mL proteinase K in IDTE buffer for 10 min at 40 C.
CRITICAL: The optimal proteinase K concentration and digestion time need to be determined for different tissue type and age. High concentration of proteinase K results in the loss of tissue. Low concentration of proteinase K results in weak signals.  CRITICAL: The two HCR hairpins need to be annealed separately.

Add both annealed hairpins into 100 mL amplification buffer (1:50 dilution).
Note: A minimum of 30 mL of diluted hairpin solution is required for one slide. For hairpin annealing, we do not recommend volume below 2 mL to avoid problem caused by evaporation in the thermocycler. The 100 mL diluted working solution prepared here from the 2 mL of stock solution is sufficient for 3 slides. If the user has more than 3 slides, the amount used need to be scaled up accordingly.
CRITICAL: The incubation should be performed in dark.
Pause point: the slides can be stored at 4 C for a month before imaging.

Timing: 2 h
This step is for imaging the samples prepared in the above steps. We will provide general guidance for this process, not specific parameters because the users likely have different microscopes and the specific optics, lasers, age of the microscopes will influence how the parameters are determined.

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performed. We encourage the users to use super resolution imaging when the density of the puncta within the cell of interest is too high to be resolved by regular confocal microscope.
CRITICAL: To facilitate quantification of foci, the user should acquire the image with sufficient resolution. This can be achieved by zooming into individual punctum and counting the number of pixels contained by the punctum. Users should avoid using the resolution below 5 pixels/mm as it is hard to distinguish individual puncta under this resolution ( Figure 5).
Note: The intensities of the brightest and weakest puncta should be within the detection range of the sensor. The users should avoid overexposing and underexposing the signals regardless of the microscope being used. In most modern fluorescent microscopes, histograms of the pixel intensities are plotted. One can adjust system gain, background threshold, and laser power to set a dynamic range that has the pixel intensity to be well distributed with few pixels beyond this range. Some software, such as Zeiss Zen, has a ''range indicator'' to show overexposed and underexposed pixels.
39. Export the images as tiff files for analysis.

QUANTIFICATION AND STATISTICAL ANALYSIS
Manual spot counting in FIJI We describe the process of counting fluorescent foci in FIJI to quantify the expression level of targeted RNA. For more information on using FIJI, see https://imagej.net/learn/. For users who do not have access to high resolution confocal microscope or super resolution microscope, one can image the experiment and control samples with an epifluorescence microscope with the same settings including excitation intensity, camera exposure time, detector gain, and filters, then quantify the average fluorescence intensity within the cells like in the traditional in situ hybridization methods whose signals are not dot-like.
1. Import the images to FIJI by using function ''File/Open.'' (Ctrl + O). Note: The user should pay attention to whether the boundaries of the puncta are split. Nonsplit puncta will be counted as one in the later steps in FIJI. The trained model can be reused.

LIMITATIONS
The probe design method in this protocol is time consuming and there is no guarantee that the probes will always generate specific and robust signals. The probes need to be tested in experiment to know their performance. This protocol is optimized for neonatal olfactory epithelium, eye, and brain tissue. We have not tested the protocol using other tissue types and at other developmental stages. The users may consider optimizing the length of fixation, the strength of proteinase treatment, and adding the methanol treatment. This protocol is developed using thin cryostat sections. Additional optimization is needed for thick sections or paraffin embedded tissue sections.

TROUBLESHOOTING Problem 1
When measuring the DNA concentration of the maxiprep, no or little DNA is detected by the nanodrop (before you begin, step 12, cc).

Potential solution
This may be caused by three reasons. First, wrong antibiotic is used during the inoculation. Use kanamycin rather than ampicillin. The culture will not look cloudy after overnight inoculation if a wrong antibiotic is used. Second, the precipitated plasmid pellet is lost during the removal of supernatant. There should be an opaque pellet after step u. When the DNA purity is high, the pellet looks translucent. The users should be careful when removing the supernatant and keep the pellet. Third, a large volume of culture growing directly from glycerol stock sometimes does not work well. Adding intermediate steps, such as streaking out colonies or growing with a smaller starter volume might also help.

Problem 2
After running the gel using the digestion product, no or little DNA is observed (before you begin, step 14, f).

Potential solution
To identify the problem, we provide several diagnosis methods and solutions to various causes of the issue.
DNA concentration is not accurately determined after the maxiprep. If there is significant among of RNA left in the prep, it can cause miscalculation of the DNA concentration and result in insufficient amount of DNA used in the digestion. The users may first check the expiration date of the P1 buffer, and whether RNase is added to it. Insufficient RNA removal can happen when the RNase in the P1 buffer has expired or not present. If this is the case, the problem can be solved by using P1 buffer containing fresh RNase and storing P1 with RNase at 4 C. The users can then measure the DNA absorption spectrum by Nanodrop. A 1.8 ratio between the absorption at 260 nm and 280 nm generally indicate good quality DNA. The users can then run the maxiprep product on a gel to examine whether contaminants exist. A strong signal around 100-200 bp area would indicate large amount of residual RNA. A strong signal around the loading vial indicates genomic DNA contamination. Second, too much DNA is added to the column. If the QIAGEN tip is overloaded, the yield can be low. To avoid adding too much DNA, less culture should be used. Third, ethanol is not added to the Zymo wash buffer. This will result in DNA being washed away from the column.

Problem 3
The digestion product forms continuous smear rather than discrete bands in the gel (before you begin, step 14, f).

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Potential solution This may be caused by loading too much DNA. The buffering capacity of sodium borate buffer is limited. When too much DNA is loaded and run in this buffer, the bands tend to elongate and become smears. This can be solved by loading the digestion product into multiple wells to reduce the DNA loaded into individual well.

Problem 4
When examine the ssDNA on the agarose gel, the bands look very bright and correspond to the sizes of possible dsDNA products resulting from partial melting. No or weak band corresponding to the size of preamplifier is observed. The band have a similar size of the undigested plasmid (before you begin, step 14, f).

Potential solution
This may be caused by two reasons. First, the sample was not fully desalted. Trace amounts of salt from NEBuffer r3.1 can inhibit the DMSO mediated melting. This can be solved by using the column based desalting method and two rounds of washing using wash buffer. Second, DMSO concentration is too low. The DMSO:sample ratio should be above 3:2.
Problem 5 ssDNA bands are observed when running the digestion product, but no ssDNA is observed in the quantification gel (before you begin, step 15, g).
Potential solution This is normally caused by agarose being not fully dissolved. DNA remains in the undissolved agarose gel and is not eluted. This can be solved by fully melting the agarose gel using more ADB buffer. Using more ADB buffer and vigorous shaking can speed up the melting.

Problem 6
Tissue disappears from the slide after overnight hybridization (step-by-step method details, step 23).

Potential solution
The proteinase K treatment may be too harsh. When this happens, one can lower the concentration of proteinase K, use a shorter incubation time, or do both. If the user wants to optimize the proteinase digestion, we recommend the user test a series of different proteinase concentration and incubation time. The user may start with 100 mg/mL, 10 mg/mL, 1 mg/mL, 0.1 mg/mL, 30 min, 10 min, and 5 min, then proceed with the conditions where the tissue does not show obvious damage, compare the end results to determine the optimal condition.

Problem 7
The hybridization solution covering the tissue is dried after overnight hybridization (step-by-step method details, step 23).

Potential solution
Enough water should be added to the bottom of the humid chamber. The users should also make sure the hydrophobic boundary is working properly. The hybridization solution should not spill outside of the boundary after being added. Other common problems are the use of expired ImmEdge pen and not waiting until the boundary is dried before adding solutions.

Problem 8
No signal is observed after the procedure (step-by-step method details, step 37). Potential solution There are many reasons that can result in failed experiment. We list a few common problems we have experienced.
Preamplifier is not successfully synthesized. The user may run the preamplifier used in the experiment on a gel similar to the one shown in Figure 2C. It is also important to examine the preamplifier in a gel at the synthesis step rather than simply measure the concentration using a Nanodrop. A high reading in the Nanodrop does not equal to high quality ssDNA. The preamplifier should be single stranded, contain 20 repeats of the HCR initiator, and of high concentration and purity. When too little or double stranded preamplifier is added to the Yn situ, no signal is developed. Another common problem is RNA degradation. It is hard to identify if this is the issue or which step this issue appears without going through complicated RNA extraction and microchip analysis of the RNA content from the tissue. In Yn situ, the RNase mediated degradation is likely coming from before step 7. The user should proceed directly to the following steps once the tissue is sectioned, mounted onto the slide and placed at room temperature. HCR hairpins are annealed in the same tube. When the HCR hairpins are heated in the same tube, they will bind to each other directly. No HCR reaction will happen on the tissue. Probe does not work. This can be identified by several diagnosis step. First, the user may check the expression pattern of the gene from literature and RNA-Seq data if it exists to make sure the gene is expressed by the tissue or cell type. Second, the user may try to perform Yn-situ using the same tissue but with a control probe. The control probe is a probe that works before, a probe designed against a highly abundant gene, or a probe designed with the same sequence used by another in situ method. It is possible for the designed probes to not bind to the target. The users can design different probes or increase the number of probe pairs to include more probes.

Problem 9
For genes with known expression pattern, signals can be seen in the regions where the gene is not expected to be expressed. Puncta are observed across the tissue with no specific patterns but are clearly distinguished from the background (step-by-step method details, step 37).

Potential solution
This may be caused by two reasons. First, too much primary probe was used. The correct concentration of primary probe in the hybridization solution is 4 nM. This concentration is for individual oligos, not for the oligo mixture, which is 40 nM when 5 pairs of probes are used. Second, the designed probe is not specific. Different probes should be designed when this is the case.

Problem 10
Specific signals are observed only in the cells or regions where the target gene is known to express. The signals are above the background but the signal to background ratio is not as good as described in the original paper. The signals are too weak to image (step-by-step method details, step 37).

Potential solution
Weak signals may result from insufficient hybridization. This can be improved by increasing the proteinase K treatment strength and adding the methanol permeabilization step. Another possibility is that the target probes do not hybridize efficiently. One can add more probes targeting to different regions of the same transcript.

Problem 11
Bright fluorescence comes from all areas in the tissue. These background signals do not appear to restrict in specific cells nor regions of the tissue. The fluorescence is not puncta-like but homogenous (step-by-step method details, step 37).