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

Abstract

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.

Figure 1:. Workflow for preparation and processing of intestinal Swiss rolls.

(A) Intestinal segments are flushed with a syringe containing PBS and then washed in PBS. (B) Wet intestinal tissue is then placed on dry filter paper. (C) The intestine is cut open longitudinally on filter paper and (D) gently splayed. (E) A piece of filter paper is laid on top of the opened intestine, and the intestine is gently sandwiched between the filter paper and stapled. The intestinal segments are fixed overnight, and (F) is rolled using forceps. (G) The intestine is gently dislodged from the reverse action forceps, and (H) has a rosette appearance. (I, J) Intestinal tissue is pinned and placed in a large cassette. (K) All four intestinal segments are placed in the same cassette for tissue processing and embedding.

Figure 2:. Hematoxylin and eosin staining of all four segments of the intestine.

(A) Tile scanning demonstrates the ability to visualize the entire intestine in a single slide with individual Swiss rolls of the duodenum, jejunum, ileum, and colon. (B) Micrograph of an intestinal Swiss roll and (C) higher magnification inset to show villi and crypt architecture. (D) Quantification of small intestine villi length in the shows no significant difference between Swiss rolled tissue and unopened tissue. Scale bars = 1000 μm.

Figure 3:. Immunofluorescence staining of all four segments of the intestine.

Adult C57BL/6J control mice were immunostained for nuclei (cyan), the lateral membrane marker, E-CADHERIN (green), goblet cells identified by MUC2 (yellow), and the apical brush border marker, γ-ACTIN, (magenta). Scale bars = 50 μm.

Figure 4.. Immunofluorescence staining of the intestine.

(A) A representative micrograph of immunofluorescence staining of the mouse intestine highlights nuclei (cyan), proliferative cells, PCNA (green), and lamina propria, LAMININ (magenta). (B) Immunofluorescence image identifying nuclei (cyan), the cell membrane marker β-CATENIN (green), and the lysosome marker LAMP1 (magenta) in the intestine. Scale bars = 50 μm.