Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue

Our research focuses on understanding how the gastrointestinal tract functions and how these functions are impacted by different pathological states. We are interested in learning the response of different intestinal epithelial cells to various conditions such as infection and inflammation, obesity, and genetic mutations. The importance of antibody specificity is becoming increasingly apparent.

However, the impact of tissue fixation, preservation, and standardized staining methods are underappreciated in the field. Consistent and robust immunofluorescent staining greatly depends on the quality of tissue preservation, processing, and orientation that occurs in the initial steps of data collection. This protocol provides a simple and effective method for investigating various questions regarding intestinal cell biology and physiology.

Our lab is currently using these protocols to better our understanding of the roles of different intestinal cell types in maintaining homeostasis and limiting inflammation. This protocol allows detailed visualization of all regions of a mouse intestine in a single paraffin-embedded block. This saves money on paraffin and paraffin-embedding services, the amount of time required for sectioning, and reagents.

It's also highly reproducible using diverse antibodies. 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 5 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 weight 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.

To begin, prepare a humidified chamber by taking a large slide box and removing the cardboard cover adhered to the lid. Place damp paper towels vertically at the base of the slide box, ensuring they lay flat. Prepare the slide for staining by deparaffinization, rehydration, antigen retrieval, and washing.

Remove the slide from PBS and wipe off excess PBS while avoiding the tissue. With a hydrophobic pen, draw a box around the tissue to form a barrier. Place the outline slide horizontally over the damp paper towels.

Add approximately 100 microliters of blocking buffer to cover the tissue. Close the humidified chamber and incubate slides at room temperature for 90 minutes. Then, gently tap off the blocking solution.

Immediately add the prepared mouse-on-mouse block to cover the tissue and incubate at room temperature for 15 minutes. At the end of the incubation, remove the slides from the humidified chamber and place them directly into a slide rack submerged in PBS. Then, remove the slides from PBS.

Gently tap off the excess PBS and place them horizontally back in the humidified chamber. Add appropriately diluted primary antibodies of interest to cover the tissue. Once the slides are removed from the humidified chamber, gently tap off the primary antibodies and place the slides in a slide rack submerged in PBS for five minutes.

After removing slides from PBS and tapping off excess PBS, place them horizontally back in the humidified chamber. Add appropriately diluted secondary antibody fluorophores or conjugated primary antibodies of interest to cover the tissue. Then, add appropriately diluted hoist directly on the tissue before incubating at room temperature in the dark.

After that, place the slides in a new solvent-resistant dish filled with PBS for five minutes in the dark. Remove one slide at a time, keeping the remaining slides submerged in PBS in the dark. After removing excess PBS, add one to two drops of antifade mounting medium to the center of the tissue.

Hold the clean cover slip by the edges and slowly lower it onto the slide at a 45 degree angle. Starting at the center of the tissue, gently press down on the cover slip with two fingers. Attach a cut, non-filtered P200 pipette to the tip of the serological pipette connected to a vacuum.

Following the edges of the cover slip, use the vacuum to remove the excess mounting medium. In a new slide box, horizontally lay the slide flat before letting it dry in the dark at room temperature. Microscopic images showed the morphology of different intestinal segments and staining for goblet cells, the apical membrane, the lateral membrane of epithelial cells, and nuclei.

Immunofluorescent staining of the intestine highlighted many different cellular compartments, including proliferative cells, interstitium, the lysosomal domain, and the epithelium.