To begin, attach a large P200 cut pipette tip to a 10-milliliter syringe and fill it with PBS. Use forceps to insert the syringe tip into the opening of an intestinal segment isolated from the euthanized mouse. Hold the intestinal segment on the syringe with the forceps and gently flush at a rate of approximately 50 microliters per second to remove the contents.
Collect the intestinal contents into an empty weigh boat. Then lay the wet intestinal segment in a straight line on a strip of dry labeled cellulose filter paper. Using dissection scissors, cut the intestine open longitudinally along the mesenteric line, approximately five millimeters at a time.
Use the bottom edge of the scissors or a pair of forceps to lay the cut tissue flat on the filter paper. Now place another piece of filter paper on top of the intestinal segment to prevent it from curling or losing its shape during fixation. To secure the tissue in place, staple the edges of the filter paper while avoiding stapling the tissue.
Submerge the tissues in 10%normal buffered formalin. Gently remove the top piece of filter paper touching the luminal side of the intestine. Use forceps to carefully peel the intestine off the bottom piece of filter paper before discarding it.
Then wash the tissue in PBS three times to remove formalin from the tissue. Using reverse action forceps, pick up a piece of intestine along the short edge with the luminal side facing up and turn the forceps to roll the tissue. Place the tissue in a dry weigh boat.
Hold the tissue in place with forceps in one hand. Carefully open the reverse action forceps and release the tissue, using the other forceps to dislodge the tissue from the reverse action forceps. Insert a size 00 dissecting pin into the tissue.
Use wire cutters to remove the sharp tip of the pin. Once all samples have been rolled, place all four tissue rolls into the same cassette and insert the cassette into a tissue processor. At an embedding station, place a small amount of paraffin into a mold.
Remove the four Swiss rolls from the cassette and place them into an individual mold, laying them as flat as possible. Add more paraffin to fill the mold. Move the mold to the cold plate and ensure that all Swiss rolls are flat on the bottom of the mold.
Place a labeled small cassette top on the mold. Once the wax has solidified, remove the block from the mold.
