The overall goal of this procedure is to visualize ICC networks of the mouse GI tract. This is accomplished by first dissecting and isolating the entire GI tract and separating it into the stomach, small intestine, cecum, and large intestine. Next, each part of the GI tract is fixed either an acetone followed by mucosal removal for whole mount imaging or in power formaldehyde, followed by embedding and cryosectioning for cross-sectional imaging.
The samples are then immuno stained against kit and other epitopes to label ICC and other relevant structures. Finally, images are required using a fluorescence microscope. Ultimately, results can be obtained that show the effects of genetic or environmental manipulations of mice on ICC architecture through immunofluorescence microscopy.
This method can help answer key questions in the ICC field, such as how specific signaling networks and transcription factors affect ICC network architectures Generally individuals to new to this method will struggle because improper stripping of the mucosal layer and orientation of the whole mount will result in significant background staining and loss of specific signal. To begin dissection of the mouse gastrointestinal tract, pin the limbs of a euthanized mouse onto a styrofoam surface and rinse the abdominal surface with 70%ethanol. Open the abdominal cavity with a long midline incision from the diaphragm to the pubis.
Make one cut at the distal esophagus and a second cut at the distal large intestine and remove the entire gastrointestinal tract from the stomach to anus on block. Carefully cutting ligaments at the duodenum and seum using small scissors. Place the gastrointestinal tract in a Petri dish with PBS.
Dissect away the mesentery using tweezers to separate the stomach, small intestine and large intestine. Cut at the pylori and ileocecal junction using a five milliliter syringe attached to a feeding needle or blunt needle. Rinse the lumen of each part of the GI tract with PBS.
Open the stomach, small intestine and large intestine along the mesenteric border. Cut two pieces for each GI organ, one for whole mount visualization and one for cryo sections. Fix the whole mount sample in acetone prior to cryosectioning to prepare the sample for immunos staining.
First, wash the sample twice in PBS. Then place the sample in a Petri dish of PBS under a dissecting microscope. Carefully scrape off the mucosal layer with a scalpel by holding one end with tweezers leaving the muscularis cut the muscle into five millimeter pieces for staining with different antibodies.
Do not store the sample for more than two days before staining in a 1.5 milliliter micro refuse tube. Incubate the sample in blocking buffer for one hour at four degrees Celsius. Next, incubate the sample in primary antibodies diluted in blocking, buffer and rotate overnight at four degrees Celsius.
We have successfully used a CK two rat anti kitt rabbit anti a O one and rabbit anti PGP 9.5 antibodies for whole mount staining. The next morning. Wash the samples twice for five minutes each in PBS with gentle rotating.
Then incubate the samples in secondary antibodies diluted in blocking buffer for two hours. At room temperature, we typically use Alexa Fluor 4 88 anti rabbit and Alexa 5 94 anti rat antibodies. Wash the samples in PB S3 times for 15 minutes each.
Using a dissecting microscope mount the samples on slides serosal side up by removing as much PBS as possible. Applying around 50 microliters of hard set mounting medium and placing a cover slip on top. Allow the mounting medium to set overnight and store the slides at minus 80 degrees Celsius.
For viewing and imaging, use a wide field objective microscope. With a Z drive, we use either a 20 times air objective or a 60 times oil objective and image, one or two micron Z sections that span the entire muscularis. The images are subsequently de convoluted using auto quant deconvolution software.
Alternatively, confocal microscopy can be used to prepare cryo sections for immuno staining. Place each part of the gastrointestinal track sal side down flat on filter paper. Cut the filter paper around the tissue.
Place the tissue on the filter paper in 4%paraldehyde in PBS for two hours at room temperature. Longer fixation times may result in antigen masking, particularly for the a K two anti kit antibody. Transfer the tissue into 30%Sucrose in PBS allow the tissue to sink overnight at four degrees Celsius.
Equilibrate the tissue with a one-to-one ratio of 30%sucrose and OCT for one hour. Then mount the tissue vertically in a cryo mold filled with OCT. The longitudinal axis of the GI tract should be in parallel with the cryo sections.
When examining mice with different genotypes or from different treatments, it may be useful to mount the tissue from different mice onto the same cryer mold. To ensure parallel processing, freeze the molds on dry ice and store at minus 80 degrees Celsius. Cut 10 micron cryo sections onto slides, placing two sections per slide and store at minus 80 degrees Celsius.
We typically perform h and d staining on one slide for immunofluorescence. First equilibrate the slides at room temperature for 15 minutes. Then draw a circle around the tissue using a pap pen.
Incubate the sections in 150 microliters of blocking buffer for one hour. Aspirate the blocking buffer and add 150 microliters of primary antibody diluted in blocking buffer and incubate overnight at four degrees Celsius in a humidified chamber. Washing PBS twice for five minutes each incubate the samples with secondary antibodies diluted in blocking buffer for two hours.
At room temperature, we typically use Alexa 4 88 anti rabbit and Alexa 5 94 anti rat antibodies. Wash the sections in PB S3 times for 15 minutes each spray it slides without completely drying the tissue. Add a drop of hard set mounting medium with DPI and place a number 1.5 cover slip on top.
Place the slides at room temperature overnight to set image the samples using dpi, fite and Texas red filter sets. Examining the large intestine from the cirr to the mucosa. The thinner layer of longitudinal muscle or LM is first encountered, followed by the thicker circular muscle or cm.
The section should be in parallel with the longitudinal muscle and perpendicular to the circular muscle, so the nuclei of LM should be somewhat elongated while the nuclei of the CM should be small and round between the LM and the CM are the myenteric plexi made of neurons that stain with PGP 9.5, PGP 9.5. Also stained neuronal processes throughout the CM examining the whole mount. As you focus from the cover slip, you'll encounter the I-C-C-M-Y-I-C-C-I-M of the CM and I-C-C-S-M-P.
The MYENTERIC ICC network surrounds the myenteric plexus and is made of multipolar cells within the longitudinal muscle layer. There are rare intramuscular, which are bipolar cells running in parallel with the muscle within the circular muscle layer. There are abundant ICC ims, which are also bipolar cells that run parallel with the circular muscle and are cross sectioned with this cut at the junction of the muscularis and submucosa.
The submucosal ICC network is a flat network of multipolar cells Once mastered. This technique can be done in three days if it is performed properly. You should have a good understanding of how to dissect and immuno stain the mouse GI tract to observe the ICC architecture.
