The overall goal of this procedure is to micro dissect two spatially distinct regions of the colon, the proliferative K crypt zone, and the differentiated surface epithelial cells. This is accomplished by first removing the mouse colon. The second step is to freeze the tissue between two metal plates.
Next, the tissue is mounted on a pre leveled OCT block. The final step is to section the tissue on a cryostat at 10 micron intervals. Ultimately, real-time PCR or Western blot is used to show changes in gene and protein expression.
The advantage of our technique is that it's fast, inexpensive, and high yield demonstrating. Our procedure is Christian Garner Schmidt, a technician in our group. All procedures using animals were reviewed and approved by the Emory University Institutional Animal Care and Use Committee, and were performed according to National Institutes of Health criteria.
After setting up the cryostat, as usual, equilibrate the cryostat blades and chucks to minus 20 degrees Celsius. Then fill an empty cryo mold with optimal cutting temperature compound and equilibrate to minus 20 degrees Celsius. This will take approximately 30 minutes.
Remove the frozen OCT from the mold and fix it to the chuck with liquid OCT. Place the chuck in the microtome arm and allow the OCT to set for 10 minutes. Set the cryostat to 10 microns per section and shave the OCT block until it is flat back the microtome arm away from the blade by several centimeters.
Next, prepare two razor blades per sample using a metal file, dull the sharp edge of the blades. Then remove the aluminum flange with forceps. Prepare a 10 centimeter tissue culture dish, 50%filled with silicon, and with 10 to 15 dissection pins.
Add five milliliters of room temperature, hanks buffer to the dish after euthanizing the mouse according to approved methods and excising a distal colon segment between zero and six centimeters from the anus. Use scissors to cut. Open the colon longitudinally and remove the fecal contents.
Pin the tissue to the silicone dissection dish containing Hank's buffer with the mucosal surface facing up. Stretch the tissue using the dissection pins. Then let it rest for 10 minutes at room temperature to remove folds from the tissue and to allow the muscle layers to relax.
Acts after the tissue has rested, remove all but the end pins. Slide a razor blade under the desired section of tissue, and then pour off the hanks buffer. Add another razor blade to the top making a sandwich.
Cut the tissue segment and remove the end pins. Transfer the razor blade tissue sandwich to dry ice. Allow the tissue to freeze for five to 10 minutes.
Pick up the razor tissue sandwich and allow it to warm slightly by placing a finger on the top blade. Separate the sandwich and cut around the edges of the tissue segment. Cutting a tissue segment approximately 0.5 centimeters by one centimeter.
Allow the tissue to warm until the ice on top of the tissue begins to melt. At this point, quickly and carefully press the tissue into the OCT block. In the cryostat place a finger over the sample.
The heat transfer will allow you to remove the blade without disrupting the tissue. Coat the tissue with OCT and allow it to equilibrate for five minutes. Then cut sections at 10 microns.
Put the sections on a slide and visually inspect the sections until the crypts are seen. Place sections in 1.5 milliliter tubes or on slides. This image shows colonic tissue stained with hematin eoin in traditional cross-section with the circular muscularis labeled cm.
The muscularis mucosa labeled MM the crypt base cells, transitional cells and surface cells are clearly seen. This image shows the circular muscularis after serial sectioning. As we proceed towards the surface cells, the muscularis mucosa is seen.
This image shows h and D stained crypt base cells at higher magnification. Note the absence of a central lumen. Here, transitional cells are seen and this image shows the surface cells.
The following images show immunofluorescent staining of isolated colonic crypts staining for the cell to cell junction protein. Soula occludin one or ZO one appears green and nuclei, which appear blue are observed in cross-section. The transitional and surface cells are shown here after serial sectioning.
Several differentiation factors are expressed in a spatiotemporal fashion along the cryp surface axis. This includes the transcription factor cripple like factor four shown in green, which is enriched in surface cell populations when viewed in cross-section serial sectioning allows real-time PCR assessment of KLF four mRNA levels between the three compartments. Finally, this western blot demonstrates levels of KLA four protein in these cell populations.
Don't forget that working with microtone blades can be extremely hazardous, so be extra careful whenever you're performing this procedure.
