TITLE: Supplementary protocols for &#39;A simple and fast optical clearing method for whole-mount fluorescence in situ hybridization (FISH) imaging&#39;
AUTHORS: Junwoo Suh, Yehe liu, Jordan Smith, Michiko Michiko Watanabe, Andrew Rollins, Michael W. Jenkins

[DESCRIPTION]
We report a single-step optical clearing method that is compatible with RNA fluorescence in situ hybridization (FISH) imaging. We previously demonstrated microscopy imaging with immunohistochemistry and genetic reporters using a technique called lipid-preserving refractive index matching for prolonged imaging depth (LIMPID). Our protocol reliably produces high-resolution 3D images with minimal aberrations using high magnification objectives, captures large field-of-view images of whole-mount tissues, and supports co-labeling with antibody and FISH probes. We also custom-designed FISH probes for quail embryos, demonstrating the ease of fabricating probes for use with less common animal models. Furthermore, we show high-quality 3D images using a conventional fluorescence microscope, without using more advanced depth sectioning instruments such as confocal or light-sheet microscopy. For broader adoption, we simplified and optimized 3D-LIMPID-FISH to minimize the barrier to entry, and we provide a detailed protocol to aid users with navigating the thick and thin of 3D microscopy.

[BEFORE_START]
To better preserve mRNA, make sure all equipment and lab surfaces are cleaned properly to prevent RNAse contamination (e.g., RNAse away, Thermo Scientific 21-402-178).

[GUIDELINES]
For figures mentioned in the protocol, please refer to the main manuscript.

[STEPS]
SECTION: Synthesis of H2O LIMPID
1. Place a 500 ml glass beaker on a large weight balance and tare. 
SECTION: Synthesis of H2O LIMPID
3. Weigh out the same weight (200 g) of   powder and gently transfer the urea powder to the glass beaker. This will make a roughly 50 Mass Percent urea solution, which will clear the sample (Supplementary Fig. 5 a-b).

SECTION: Synthesis of H2O LIMPID
2. Transfer 200 g of deionized   to the glass beaker. Remove the beaker from the balance.

SECTION: Synthesis of H2O LIMPID
4. Add a magnetic stirring bar in the beaker.
SECTION: Synthesis of H2O LIMPID
5. Wrap the top of the beaker to reduce water evaporation.
SECTION: Synthesis of H2O LIMPID
6. Heat up the beaker to 60 °C and gently mix the solution with the stirring bar until all powder is dissolved (becomes transparent when fully dissolved).

SECTION: Synthesis of H2O LIMPID
7. Once all urea powder is dissolved, cool down the solution and transfer 300 g of 50% (w/w) urea solution to an empty 500 ml glass beaker.

SECTION: Synthesis of H2O LIMPID
7.1. Place the empty glass beaker on the large weight balance and slowly pour 50% (w/w) urea solution until 300 g of solution is transferred.

SECTION: Synthesis of H2O LIMPID
7.2. Transfer remaining 50% (w/w) urea solution to a glass container with a cap. Close the cap and reduce moisture loss by wrapping the cap with a parafilm.
SECTION: Synthesis of H2O LIMPID
7.3. Transfer the magnetic stirring bar to the new beaker once all liquids are transferred.
SECTION: Synthesis of H2O LIMPID
8. Weigh out 200 g of   powder on a weighing boat.

SECTION: Synthesis of H2O LIMPID
9. Gently transfer the Nycodenz powder to the glass beaker. The weight ratio of iohexol to urea solution is now 2:3

SECTION: Synthesis of H2O LIMPID
10. Wrap the top of the beaker with parafilm to reduce water evaporation.
SECTION: Synthesis of H2O LIMPID
11. Heat up the beaker to 60 °C and gently mix the solution with the stirring bar until all powder is dissolved.

SECTION: Synthesis of H2O LIMPID
11.1. Nicodenz can take a long time (3-6 hours) to dissolve.
SECTION: Synthesis of H2O LIMPID
11.2. The solution can be left overnight if it is not heated (room temperature) and the beaker is well covered with parafilm.
SECTION: Synthesis of H2O LIMPID
12. Take a small sample (~50 µL) from the beaker and measure the refractive index with an abbe refractometer.
 


SECTION: Synthesis of H2O LIMPID
13. In order to adjust the refractive index of the LIMPID solution, add Nycodenz powder or 50% (w/w) urea solution. Use the calibration curve (supplementary figure 1) for rough estimates.
SECTION: Synthesis of H2O LIMPID
13.1. Nycodenz will increase the refractive index (up to ~1.57).
SECTION: Synthesis of H2O LIMPID
13.2. 50% urea solution will decrease the refractive index (down to ~1.41).
SECTION: Synthesis of SSC-LIMPID
15. Prepare 200 ml of SSC buffer by diluting x ml of   buffer with y ml of deionized  .

SECTION: Synthesis of SSC-LIMPID
15.1. For 5xSSC-LIMPID: x = 50 ml, y = 150 ml
SECTION: Synthesis of SSC-LIMPID
15.2. For 2xSSC-LIMPID: x = 20 ml, y = 180 ml
SECTION: Synthesis of SSC-LIMPID
16. Place a 500 ml glass beaker on a large weight balance and tare.
SECTION: Synthesis of SSC-LIMPID
17. Transfer 200 g of SSC buffer to the glass beaker. Remove the beaker from the balance.
SECTION: Synthesis of SSC-LIMPID
18. Weigh out the same weight (200 g) of   powder and gently transfer the urea powder to the glass beaker. This will make a roughly 50 Mass Percent urea solution, which will clear the sample (Supplementary Fig. 5 a-b).
SECTION: Synthesis of SSC-LIMPID
19. Add a magnetic stirring bar in the beaker.
SECTION: Synthesis of SSC-LIMPID
20. Wrap the top of the beaker to reduce water evaporation.
SECTION: Synthesis of SSC-LIMPID
21. Heat up the beaker to 60 °C and gently mix the solution with a stirring bar until all powder is dissolved.
SECTION: Synthesis of SSC-LIMPID
22. Once all urea powder is dissolved, cool down the solution and transfer 300 g of 50% (w/w) urea solution to an empty 500 ml glass beaker.
SECTION: Synthesis of SSC-LIMPID
22.1. Place the empty glass beaker on the large weight balance and slowly pour 50% (w/w) urea + SSC solution until 300 g is transferred.
SECTION: Synthesis of SSC-LIMPID
22.2. Transfer remaining 50% (w/w) urea + SSC solution to a glass container with a cap. Close the cap and reduce moisture loss by wrapping the cap with parafilm.
SECTION: Synthesis of SSC-LIMPID
22.3. Transfer the magnetic stirring bar to the new beaker once all liquids are transferred.
SECTION: Synthesis of SSC-LIMPID
23. Weigh out 200 g of  powder on a weighing boat.
SECTION: Synthesis of SSC-LIMPID
24. Gently transfer the Nycodenz powder to the glass beaker. The weight ratio of iohexol to urea solution is now 2:3.
SECTION: Synthesis of SSC-LIMPID
25. Wrap the top of the beaker with parafilm to reduce water evaporation.
SECTION: Synthesis of SSC-LIMPID
26. Heat up the beaker to 60 °C and gently mix the solution with the stirring bar until all powder is dissolved.

SECTION: Synthesis of SSC-LIMPID
26.1. Nicodenz can take a long time (3 - 6 hours) to dissolve.
SECTION: Synthesis of SSC-LIMPID
26.2. The solution can be left overnight if it is not heated (room temperature) and the beaker is well covered with parafilm.
SECTION: Synthesis of SSC-LIMPID
27. Take a small sample (~50 μl) from the beaker and measure the refractive index with an Abbe refractometer.
 

SECTION: Synthesis of SSC-LIMPID
28. In order to adjust the refractive index of the SSC-LIMPID, add Nycodenz powder or 50% (w/w) urea solution. Use the calibration curve (supplementary figure 1) for rough estimates.
SECTION: Synthesis of SSC-LIMPID
28.1. Nycodenz will increase the refractive index (up to ~1.57).
SECTION: Synthesis of SSC-LIMPID
28.2. 50% (w/w) urea + SSC solution will decrease the refractive index (down to ~1.41).
SECTION: Methanol dehydration/delipidation
30. Prepare 4 % (v/v) paraformaldehyde (PFA) solution with 10 mL of 16 % (v/v)  , 4 mL of   and 26 mL of deionized water.

SECTION: Methanol dehydration/delipidation
31. Fix fresh samples in PFA solution at 4 °C overnight.

SECTION: Methanol dehydration/delipidation
32. Wash sample 3 x 5 minutes with 1xPBST.
SECTION: Methanol dehydration/delipidation
33. Remove all fluids and immerse the sample in increasingly concentrated  solution (2 mL).

SECTION: Methanol dehydration/delipidation
33.1. 10 minutes in 25% methanol / 75% 1xPBST solution
SECTION: Methanol dehydration/delipidation
33.2. 10 minutes in 50% methanol / 50% 1xPBST solution
SECTION: Methanol dehydration/delipidation
33.3. 10 minutes in 75% methanol / 25% 1xPBST solution
SECTION: Methanol dehydration/delipidation
33.4. 10 minutes in 100% methanol
SECTION: Methanol dehydration/delipidation
34. Wash the sample with methanol for 10 minutes.
SECTION: Methanol dehydration/delipidation
35. Incubate at Room temperature overnight in methanol and the sample will become delipidated. 

SECTION: Methanol dehydration/delipidation
36. Wash the sample with methanol for 10 minutes. (Sample can be stored at -20 °C for long term storage)

SECTION: Methanol dehydration/delipidation
37. Rehydrate sample by washing it with increasingly concentrated 1xPBST solutions
SECTION: Methanol dehydration/delipidation
37.1. 10 minutes in 75% methanol / 25% 1xPBST solution on ice 

SECTION: Methanol dehydration/delipidation
37.2. 10 minutes in 50% methanol / 50% 1xPBST solution on ice

SECTION: Methanol dehydration/delipidation
37.3. 10 minutes in 25% methanol / 75% 1xPBST solution on ice

SECTION: Methanol dehydration/delipidation
37.4. 10 minutes in 100% 1xPBST on ice

SECTION: Methanol dehydration/delipidation
38. Wash the sample with 1xPBST for 10 minutes on ice

SECTION: Sample Bleaching
39. Prepare bleaching solution. For 10ml of bleaching solution: 1 mL of 5x SSC, 50 µL of  , 40 µL of , 8.91 mL of deionized H2O.

SECTION: Sample Bleaching
40. Incubate sample in the bleaching solution at Room temperature for 2 hours

SECTION: Sample Bleaching
41. Wash the sample twice with 1xPBST for 10 minutes
SECTION: Methanol dehydration/delipidation
31.1. Fixation condition and time will differ by sample size and properties. This protocol is optimized for pre-processing day 5 quail. Mouse brain was fixed with a perfusion fixation protocol, and trigeminal ganglia was fixed for 3 hours at 4 °C. 
SECTION: Sample Bleaching
40.1. A bright white light source can be added to increase bleaching.
SECTION: Air bubble removal
42. Boil 75 mLof 1xPBS.

SECTION: Synthesis of H2O LIMPID
14. If you see crystalized urea in any steps of this section, it is a sign that too much evaporation has occurred and the urea concentration is higher than 50 Mass Percent. In such a case, use a 500 mL glass container with a cap instead of a beaker. Use a powder funnel to minimize the loss and heat up the container no higher than 40 °C. 

SECTION: Synthesis of SSC-LIMPID
29. If you see crystalized urea in any steps of this section, it is a sign that too much evaporation has occurred and the urea concentration is higher than 50 Mass Percent. In such a case, use a 500 mL glass container with a cap instead of a beaker. Use a powder funnel to minimize the loss and heat up the container no higher than 40 °C. 
SECTION: Air bubble removal
43. Once the solution is boiling, stop the heating and cool it down with ice. It is important to not disturb the solution. Move it carefully to the ice bucket.
SECTION: Air bubble removal
44. Use an infrared thermometer gun to indirectly measure the temperature.
SECTION: Air bubble removal
45. Once the solution has cooled down to 36-40 °C, carefully transfer it to a 50 mL plastic tube. Fill the tube up to the top. It is important to completely fill the tube without any air pockets.

SECTION: Air bubble removal
46. Transfer the sample to the tube.
SECTION: Air bubble removal
47. Close the cap and wrap it with parafilm
SECTION: Air bubble removal
48. Cool down the sample further to 4 °C

SECTION: Air bubble removal
49. Visually check the bubbles inside the sample. If the bubbles are not completely removed, repeat step 1-7 until all bubbles are removed (Supplementary Fig. 7 b).
SECTION: HCR hybridization and amplification for large samples with cavities
50. Prepare formamide solution
SECTION: HCR hybridization and amplification for large samples with cavities
50.1. For 50%: 5 mL of formamide, 4 mL of deionized water and 1 mL of 20xSSC

SECTION: HCR hybridization and amplification for large samples with cavities
50.3. For 15%: 1.5 mL of formamide, 7.5 mL of deionized water and 1 mL of 20xSSC.

SECTION: HCR hybridization and amplification for large samples with cavities
50.2. For 30%: 3 mL of formamide, 6 mL of deionized water and 1 mL of 20xSSC.

SECTION: HCR hybridization and amplification for large samples with cavities
51. Incubate sample in 2 mL of 15% formamide solution at 37 °C for 30 minutes

SECTION: HCR hybridization and amplification for large samples with cavities
51.1. Take extra precaution on the cavity. Revert back to a lower concentration solution if the cavity is or about to collapse. Smaller increments of concentrations are recommended to alleviate osmotic pressure.
SECTION: HCR hybridization and amplification for large samples with cavities
52. Replace the 15% formamide solution with 2 mLof 30% formamide solution and incubate at 37 °C for 30 minutes

SECTION: HCR hybridization and amplification for large samples with cavities
53. Replace the 30% formamide solution with 2 mL of 50% formamide solution and incubate at 37 °C for 3 hours

SECTION: HCR hybridization and amplification for large samples with cavities
54. Thaw 100%   from the -20 °C storage and prepare 33% and 66% hybridization buffers
SECTION: HCR hybridization and amplification for large samples with cavities
54.1. For 33%: 1 mL of hybridization solution, 2 mL of 5xSSC buffer.
SECTION: HCR hybridization and amplification for large samples with cavities
54.2. For 66%: 2 mL of hybridization solution, 1 mL of 5xSSC buffer.
SECTION: HCR hybridization and amplification for large samples with cavities
55. Incubate sample in 2 mL of 33% hybridization buffer at 37 °C for 30 minutes
SECTION: HCR hybridization and amplification for large samples with cavities
56. Replace 33% hybridization buffer with 2 mL of 66% hybridization buffer and incubate at 37 °C for 30 minute
SECTION: HCR hybridization and amplification for large samples with cavities
57. Replace 66% hybridization buffer with 2 mL of 100% hybridization buffer and incubate at 37 °C overnight
SECTION: HCR hybridization and amplification for large samples with cavities
55.1. Take extra precaution on the cavity. Revert back to a lower concentration solution if the cavity is or about to collapse. Smaller increments of concentrations are recommended to alleviate osmotic pressure.
SECTION: HCR hybridization and amplification for large samples with cavities
58. Prepare a hybridization solution by mixing x pmol of hybridization probes and y ml of hybridization buffer.
SECTION: HCR hybridization and amplification for large samples with cavities
58.1. For a small sample, use the recommended recipe from the paper:
x = 2, y = 0.5.
SECTION: HCR hybridization and amplification for large samples with cavities
58.2. For larger samples, increase volume of hybridization buffer and increase the moles of probes proportionally. (e.g., x = 4, y = 1 or x = 6, y = 1.5)
SECTION: HCR hybridization and amplification for large samples with cavities
58.3. For a reference, we used 1 mL of hybridization buffer (4 pmols of probes) for the day 5 quail (~ 1 cm and larger) sample.

SECTION: HCR hybridization and amplification for large samples with cavities
59. Transfer the sample to the prepared hybridization solution.
SECTION: HCR hybridization and amplification for large samples with cavities
60. Incubate the sample in the hybridization solution at 37 °C for 2 days. Rotate or shake samples for better staining.
SECTION: HCR hybridization and amplification for large samples with cavities
61. Thaw   from -20 °C storage

SECTION: HCR hybridization and amplification for large samples with cavities
62. Wash sample with 2 mL of wash buffer at 37 °C for 4x15 minutes. Rotate or shake samples.

SECTION: HCR hybridization and amplification for large samples with cavities
63. Wash the sample twice with 2 mL of 5xSSCT for 10 minutes.

SECTION: HCR hybridization and amplification for large samples with cavities
64. Incubate sample in 2 mL of   for 30 minutes. 
SECTION: HCR hybridization and amplification for large samples with cavities
65. Prepare amplification probes in 600 µL tubes

SECTION: HCR hybridization and amplification for large samples with cavities
65.1. For small samples, use the recommended recipe from the paper:
30 pmols of probes for 500 µL of amplification buffer.

SECTION: HCR hybridization and amplification for large samples with cavities
65.2. For larger samples, increase volume of hybridization buffer and increase the moles of probes proportionally.
SECTION: HCR hybridization and amplification for large samples with cavities
65.3. For a reference, we used 1 mL of amplification buffer. 48 pmols of probes were sufficient to give a strong signal.

SECTION: HCR hybridization and amplification for large samples with cavities
66. Make snap-cool hairpins by running a heat-block program (Techne TC-3000 PCR Thermal Cycler) on the probes
SECTION: HCR hybridization and amplification for large samples with cavities
66.1. 90 seconds at 95 °C

SECTION: HCR hybridization and amplification for large samples with cavities
66.2. 30 minutes at 25 °C

SECTION: HCR hybridization and amplification for large samples with cavities
67. Spin down the 600 µL tubes with a centrifuge.

SECTION: HCR hybridization and amplification for large samples with cavities
68. Prepare an amplification solution by transferring all probes to the fresh 1 mL amplification buffer.

SECTION: HCR hybridization and amplification for large samples with cavities
69. Transfer the sample to the amplification solution.
SECTION: HCR hybridization and amplification for large samples with cavities
70. Wrap the sample container with aluminum foil and incubate it at 4 °C overnight. Rotate or shake the sample.

SECTION: HCR hybridization and amplification for large samples with cavities
71. After the overnight incubation, warm up the sample to Room temperature and incubate for 2 hours. Rotate or shake the sample.

SECTION: HCR hybridization and amplification for large samples with cavities
72. Wash the sample twice with 2 mL of 5xSSCT for 10 minutes.

SECTION: HCR hybridization and amplification for large samples with cavities
73. If desired, stain the sample with a   stain

SECTION: HCR hybridization and amplification for large samples with cavities
73.1. Prepare Dapi staining solution: 1:1000 dilution of 300 micromolar (µM) Dapi in 2 mL of 5xSSCT.
SECTION: HCR hybridization and amplification for large samples with cavities
73.2. Incubate the sample in Dapi at Room temperature overnight.
SECTION: HCR hybridization and amplification for large samples with cavities
73.3. Wash the sample twice with 2 mL of 5xSSCT for 10 minutes.
SECTION: HCR hybridization and amplification for large samples with cavities
74. If desired, the sample can be stained with antibodies
SECTION: HCR hybridization and amplification for large samples with cavities
74.1. Prepare antibody staining solution by following the manufacturer recommended dilution. E.g., 1:20 dilution of antibody in a blocking buffer.
SECTION: HCR hybridization and amplification for large samples with cavities
74.2. Block sample in 1xPBS buffer with 1.5% bovine serum albumin (BSA), 0.1% tween at 4 °C for overnight.

SECTION: HCR hybridization and amplification for large samples with cavities
74.3. Stain in antibody staining solution following manufacturer’s suggested incubation temperature.
SECTION: Tissue clearing
75. Remove as much fluids from the sample as possible.
SECTION: Tissue clearing
75.1. It is recommended that no strong force is exerted on the sample during fluid aspiration.
SECTION: Tissue clearing
75.2. It is acceptable to leave some fluids and preserve the sample integrity.
SECTION: Tissue clearing
76. Transfer LIMPID (H2O or SSC variant) to the sample-holding container.
SECTION: Tissue clearing
76.1. The volume of added LIMPID is recommended to be at least 3 times the volume of the sample.
SECTION: Tissue clearing
77. Mix the solution by placing the sample container on a rotator or shaker for 5 minutes. 
SECTION: Tissue clearing
78. Remove LIMPID and transfer fresh LIMPID to the sample container.
SECTION: Tissue clearing
79. Mix the solution by placing the sample container on a rotator or shaker for 5 minutes.
SECTION: Tissue clearing
80. Examine the sample and its transparency
SECTION: Tissue clearing
80.1. It is expected that a thick sample is not completely cleared.
SECTION: Tissue clearing
80.2. Samples with chromophores (e.g., blood) will be tinted with the color. If needed, please refer to sample bleaching section above to bleach the sample. 
SECTION: Tissue clearing
80.3. Samples with some visible opaqueness can still generate good optically sectioned 3D volumes.
SECTION: Tissue clearing
81. If more clearing is desired, remove the LIMPID and transfer fresh LIMPID to the sample container. Incubate for 15 minutes on a rotator or shaker. 
SECTION: Tissue clearing
82. Examine the sample and its transparency.
SECTION: Tissue clearing
83. If more clearing is desired, remove the LIMPID and transfer fresh LIMPID to the sample container. Incubate for 30 minutes on a rotator or shaker at 36 °C. 
SECTION: Tissue clearing
83.1. Further incubation can increase transparency of the tissue but with diminishing returns. 
SECTION: Tissue clearing
83.2. The transparency is expected to max out at 1 hour incubation at 36 °C. This can vary greatly depending on the sample.

SECTION: Tissue clearing
83.3. Note that long incubation may lead to probe leakage especially when not using SSC-LIMPID.
SECTION: Tissue clearing
84. Transfer the sample to a chambered tissue slide. A metal slide (Supplementary Fig. 6 a, method) was used for our samples.
SECTION: Tissue clearing
85. Add LIMPID to slightly overfill the chamber (Supplementary Fig. 6 a).
SECTION: Tissue clearing
86. Place a coverslip on top of the chamber.
SECTION: Tissue clearing
87. Gently push the coverslip with a round tip tweezer or a tweezer with rubber tip to squeeze out excess LIMPID from the chamber.
SECTION: Tissue clearing
88. Gently remove excess LIMPID from the slide with a Kimwipe.
SECTION: Tissue clearing
88.1. Localized evaporation of LIMPID on the edge of the coverslip will create sealing of the chamber (Supplementary Fig. 6 b) that can easily be broken later.
SECTION: Tissue clearing
88.2. Small magnets can be used to secure the coverslip (Supplementary Fig. 6 b).
SECTION: Tissue clearing
89. The sample is ready for imaging.