TITLE: Human colon tissue clearing and Immunohistochemistry
AUTHORS: Robert Heuckeroth, Silvia Huerta Lopez, Kahleb Graham, Rajarshi Sengupta

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">INTRODUCTION: Bowel pathology is routinely evaluated by sectioning tissue, staining, and light microscopy. Tissue sectioning does not provide robust information about three-dimensional structures. Defining bowel anatomy in three dimensions is especially valuable for understanding human bowel motility disorders. Intrinsic mechanisms controlling bowel motility include the enteric nervous system (ENS), pacemaker cells called interstitial cells of Cajal, smooth muscle cells and PDGFRalpha+ cells. One problem is that the cells that control motility are distributed throughout the bowel wall and the bowel wall is too thick and opaque to permit direct visualization without sectioning.</div><div class = "text-block">OBJECTIVES: To develop an efficient and reproducible method to make human colon translucent, stain with antibodies, and visualize cells that control bowel motility in three dimensions.</div><div class = "text-block"><span>METHODS: Tissue is cleaned, trimmed to remove fat, pinned flat, gently stretched and then fixed in 4% paraformaldehyde before storage. To begin clearing tissue is treated with 100% methanol, then permeabilized with Dent's bleach (Methanol, DMSO and hydrogen peroxide) and rinsed with PBS. Blocking is performed for 3 days at room temperature. Incubation with primary antibodies occurs for 14 days at 37 </span><span style = "vertical-align:super;">o</span><span>C. Unbound primary antibody is washed out over the course of one day. Secondary antibody staining is performed for three days at 37 </span><span style = "vertical-align:super;">o</span><span>C. After washing in PBS, tissue is dehydrated using a graded methanol series and then cleared using Murray's Clear (Benzyl Benzoate: Benzyl Alcohol). For confocal imaging, tissue is mounted in Murray's Clear.</span></div><div class = "text-block">RESULTS: This approach provides efficient tissue clearing and reproducible staining with a subset of tested antibodies. We routinely obtain images using two or three antibodies and can visualize stained cells all the way through the bowel wall thickness. Confocal imaging permits excellent visualization and three dimensional reconstruction. We routinely stain 1 cm x 1 cm human colon pieces, but the same methods can be used to stain and image colon tissue many centimeters in length.</div><div class = "text-block">CONCLUSION: We established an efficient and reproducible method for clearing, staining with antibodies and imaging colon tissue. This approach works well to generate three dimensional images of stained cells. We can visualize many cell types that control bowel motility, but the approach will also probably work to see other cell types with appropriate antibodies. Only a subset of antibodies work with this organic solvent based fixation and clearing method.</div></div>

[STEPS]
?. [Fresh tissue processing and fixation]
Transport fresh tissue from human colon in cold sterile 1X phosphate buffered saline (PBS) on ice. Tissues placed in cold PBS within 1-2 hours of resection were used for imaging.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Transfer tissue into Blocking Solution (4% Normal Donkey Serum in 0.5%  PBS-T (0.5%TritonX100 in Phosphate Buffered Saline with 0.05% Sodium Azide)) for 3 days at room temperature.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Incubate tissue in primary antibody diluted in Blocking Solution (4% Normal Donkey Serum in 0.5%  PBS-T (0.5%TritonX100 in Phosphate Buffered Saline with 0.05% Sodium Azide)) for 14 days at 37C. Be sure to place samples in air-tight sealed tubes to prevent contamination.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Wash tissue three times in 1X PBS with 0.05% Sodium Azide for 2 hours per wash and leave tissue in 1X PBS with 0.05% Sodium Azide overnight at room temperature on the rocker.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Incubate in Secondary Antibody diluted in 1X PBS with 0.05% Sodium Azide for 3 days at 37C.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Wash tissue three times in 1X PBS with 0.05% Sodium Azide for 2 hours per wash and leave tissue in 1X PBS with 0.05% Sodium Azide overnight at room temperature on the rocker.C. Dehydration and ClearingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Dehydrate tissue following methanol series:a.	50% methanol for 30 minutesb.	70% methanol for 30 minutesc.	80% methanol for 30 minutesd.	95% methanol for 30 minutese.	100% methanol for 30 minutesf.	100% methanol for 30 minutesg.	100% methanol for 30 minutes
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Transfer tissue directly out of 100% methanol into Murray’s Clear (2:1 Benzyl Benzoate: Benzyl Alcohol) before imaging. Tissue clearing will depend on the thickness of your specimen.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Once the tissue has become completely transparent, mount on glass slides using Murray’s Clear as your mounting media.D. Image AcquisitionImages can be acquired using confocal or light sheet microscopy. In our protocol images were acquired using an LSM 750 Confocal Microscope.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Z-stacks through the myenteric and submucosal plexus can be acquired using the 10X objective and the stitching feature on Zen software.
?. [B. ImmunostainingThe following incubations are done in 24-well plates with approximately 500ul of solution or until tissue is covered and placed on a rocker (moderate speed) at room temperature.]
Detailed volumetric and quantification of neuronal subtypes can be obtained from high-resolution images using the 20X Objective. Due to the limited working distance allowed with this objective the myenteric and submucosal plexus must be imaged independently by turning the tissue over.
?. [Fresh tissue processing and fixation]
Transfer tissue using forceps and fresh PBS into SylgardTM coated dish. Lay tissue flat with the luminal side down and the serosa side up.
?. [Fresh tissue processing and fixation]
Using dissecting scissors and forceps remove any fat tissue that is present and discard according to Biosafety instructions.
?. [Fresh tissue processing and fixation]
Pin one side of the tissue and begin to stretch and pin along the edge of the tissue until the tissue looks uniform and as flat as possible. Pins are then repositioned several times, with each round of repositioning stretching the tissue further as colon relaxes. On average, colon area stretched to about 2.6x more than the original unstretched dimensions.
?. [Fresh tissue processing and fixation]
Remove the PBS and add 4% Paraformaldehyde (PFA). Let issue sit at 4C overnight.
?. [Fresh tissue processing and fixation]
Using small dissecting scissors cut out a full thickness strip of tissue (ex. 1cm x 1 cm) to be used for staining. The remaining tissue can be transferred to a solution of (50% 1X PBS, 50% Glycerol, 0.05% Sodium Azide) and stored at 4C for future use.
?. [Immunostaining]
The following incubations are done in 24-well plates (2 cm diameter/well) with approximately 500ul of solution. Additional solution is added if needed to cover tissue. Samples are placed on a rocker (moderate speed) at room temperature for these steps.For tissue taken out of storage, wash three times in 1X PBS for 5 minutes each wash.
?. [Immunostaining]
Incubate tissue in 100% Methanol for 1 hour on ice.
?. [Immunostaining]
Transfer to permeabilization solution (Dent’s Bleach: 5mL 30% hydrogen peroxide, 5mL DMSO, 20mL 100% Methanol) for 2 hours at room temperature.
?. [Immunostaining]
Wash tissue three times in 1X PBS for 5 minutes each wash.
?. [Immunostaining]
The following incubations are done in 2.0mL Eppendorf tubes with ~500uL- 1mL of solution on a shaker at 40 rpm-100 rpm at 37C. Tissue should be completely covered with incubation solution.Transfer tissue into Blocking Solution (4% Normal Donkey Serum in 0.5% PBS-T (0.5%TritonX100 in 1X Phosphate Buffered Saline with 0.05% Sodium Azide)) for 3 days at room temperature.
?. The following incubations are done in 2.0mL Eppendorf tubes with ~500uL- 1mL of solution on the shaker at 40 rpm-100 rpm at 37C.
?. [Fresh tissue processing and fixation]
Replace the 4% PFA with fresh PBS, and begin to carefully remove the pins. Trim the edges of the tissue.
?. [Immunostaining]
Incubate tissue in primary antibody diluted in Blocking Solution (4% Normal Donkey Serum in 0.5% PBS-T (0.5%Triton X-100 in Phosphate Buffered Saline with 0.05% Sodium Azide)) for 14 days at 37C. Be sure to place samples in air-tight sealed tubes to prevent contamination. Tubes are additionally sealed with ParafilmTM.
?. [Immunostaining]
Wash tissue three times in 1X PBS with 0.05% Sodium Azide for 2 hours per wash and leave tissue in 1X PBS with 0.05% Sodium Azide overnight at room temperature on the rocker.
?. [Immunostaining]
Incubate in Secondary Antibody diluted in 1X PBS with 0.05% Sodium Azide for 3 days at 37C.
?. [Immunostaining]
Wash tissue three times in 1X PBS with 0.05% Sodium Azide for 2 hours per wash and leave tissue in 1X PBS with 0.05% Sodium Azide overnight at room temperature on the rocker.
?. [Dehydration and clearing]
The following incubations are done in 24-well plates with approximately 500ul of solution (with added liquid if needed to cover tissue. Incubations occur on a rocker (moderate speed) at room temperature.Dehydrate tissue following methanol series: 50% methanol for 30 minutes, 70% methanol for 30 minutes, 80% methanol for 30 minutes, 95% methanol for 30 minutes, 100% methanol for 30 minutes, 100% methanol for 30 minutes, 100% methanol for 30 minutes.
?. [Dehydration and clearing]
Transfer tissue directly out of 100% methanol into Murray’s Clear (2:1 Benzyl Benzoate: Benzyl Alcohol) before imaging. Tissue clearing will depend on the thickness of your specimen.
?. [Dehydration and clearing]
Once the tissue has become completely transparent, mount on glass slides using Murray’s Clear as your mounting media (if imagining on inverted Confocal microscope a coverslip can be taped at the edges of the slide to prevent it from falling off with only a minimal amount of Murray’s Clear).
?. [Image aquisition]
Images can be acquired using confocal or light sheet microscopy. We acquired images using an LSM 710 Confocal microscope. Details about our microscope inclue:-10x and 20x Plan-Apochromat objectives -Zeiss Zen software (version 2.3 14.0.14.201)) -Z-axis increments were 4μm (10x objective) or 1μm (20x objective)-Each image slice was 900x900 (10x) or 1200x1200 pixels (20x). -10x Z-stacks were stitched to cover large regions. -Laser-scanning operated under multi-track to sequentially acquire multi-channel images. -Each channel used 100% laser power. -Excitation/long-pass emission filters: Alexa Fluor® 647 (excitation: 633 nm, emission: 656-755-nm filter), AlexaFluor® 594 (excitation: 561 nm, emission: 588-656-nm filter), Alexa Fluor® 488 (excitation: 488nm, emission: 493-584-nm filter)
?. [Image aquisition]
Z-stacks through the myenteric and submucosal plexus can be acquired using the 10X objective and the stitching feature on Zen software.
?. [Image aquisition]
Detailed volumetric and quantification of neuronal subtypes can be obtained from high-resolution images using the 20X Objective. Due to the limited working distance allowed with this objective, the myenteric and submucosal plexus must be imaged independently by turning the tissue over.
?. [Image aquisition]