The intestine is vital for digestion and absorption. Each region-duodenum, jejunum, ileum, colon-serves distinct functions due to unique cellular structures. Studying intestinal physiology demands meticulous tissue analysis. This protocol outlines tissue fixation and processing using the Swiss roll technique, ensuring accurate immunostaining through proper tissue preservation and orientation.
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.
The cellular architecture of the intestine poses a unique challenge in maintaining its structural integrity when intestinal tissue is being preserved for immunostaining. The small intestine is made up of elongated fingerlike structures known as villi1. These villi often become malformed during embedding processes. Ensuring that researchers have techniques for properly embedding intestines to achieve cross sections, allowing visualization of all regions of the intestine, as well as the layers that make up the intestine (i.e., muscularis propria, mucosa, and the serosa), is crucial for robust experimental analysis2. Inadequate fixation, excessive fixation, and improper tissue handling will compromise tissue integrity, resulting in inadvertent damage to the intestinal epithelium3,4. Damaging the intestinal epithelium during these steps can significantly diminish the quality of subsequent analyses, like immunofluorescence, irrespective of the efficacy of immunohistochemistry protocols and antibodies employed.
Immunostaining, like proper tissue fixation, is an important part of biomedical research. When done well, immunostaining can illuminate previously unknown aspects of cellular structure and function. Immunofluorescence staining of paraffin sections can be challenging due to physicochemical modifications resulting from the fixation and paraffin embedding process5. Fixation and paraffin embedding results in antigen masking that can interfere with immunofluorescence detection of epitopes of interest6. Delayed fixation can induce proteolytic degradation, which results in weakened or absent staining of critical epitopes7. Additionally, antibodies are often inaccurate with high levels of background. Immunostaining protocols that promote consistent and specific antibody binding and a high signal-to-noise ratio can provide valuable information for researchers.
Here, we provide a comprehensive protocol designed to obtain high-quality immunofluorescence images through intestinal tissue fixation, Swiss roll preparation8, and immunostaining. Emphasizing guidelines to preserve the integrity of the intestine, the protocol aims to provide researchers with a robust methodology to enhance the quality and reliability of immunofluorescence imaging studies. We have also sought to use cost-effective resources, including filter paper and homemade antigen retrieval, blocking solutions, and antibody diluents to make the protocol more accessible to labs that may have restricted funds. As for all experimental protocols, researchers should optimize the current protocol based on their experimental approach and areas of interest.
The Institutional Animal Care and Use Committee of the Medical University of South Carolina approved all animal care, maintenance, and treatment. Intestinal tissue was collected from adult C57BL/6J mice (males and females 3-5 months old, weighing about 30 g) for use in the present study.
1. Intestinal tissue fixation
2. Intestinal tissue rolling and processing
CAUTION: Perform the steps 2.1-2.6 in a ventilated hood.
3. Embedding intestinal tissue
4. Tissue adherence and slide preparation
5. Deparaffinization
6. Rehydration
7. Antigen retrieval
8. Blocking nonspecific background staining
9. Mouse on mouse blocking
10. Primary antibodies
11. Washing the slides
12. Secondary antibodies and nuclei counterstaining
13. Mounting and preparation for microscopy
Hematoxylin and eosin (H&E) staining was performed, as previously described12. Using the optimized method, intestinal Swiss rolls included all three segments of the small intestine and the large intestine on a single slide. Having the entire intestine accommodated on a slide allows researchers to analyze changes throughout all portions of the intestine and saves costs on sectioning and staining reagents (Figure 1). Also, exposing all intestinal segments to the same solutions simultaneously when immunostaining helps ensure accurate results. The H&E micrograph demonstrates the preserved intestinal architecture of all portions of the small intestine and large intestine (Figure 2). Measurement of duodenal villi showed no significant difference in villi height in unopened intestinal tissue compared to this Swiss roll method suggesting that opening the intestine does not disrupt the tissue architecture (Figure 2D). Immunostaining of intestinal Swiss rolls shows the various layers of the intestine as well as the entire villous crypt axis. The fluorescent images show low background levels from the primary and secondary antibody staining and clearly depict the individual cells present in the epithelium. Figure 3 shows the morphology of different intestinal segments and staining for goblet cells (MUC2 positive cells)13, the apical membrane (γ-ACTIN)14, the lateral membrane of epithelial cells (E-CADHERIN)15, and nuclei. This protocol is suitable for identifying many different cellular compartments, including proliferative cells (PCNA)16, interstitium (LAMININ; Figure 4A)17, the lysosomal domain (LAMP1)14, and the epithelium (β-CATENIN; Figure 4B)18.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Here, we present an optimized method for tissue fixation using the Swiss roll technique to preserve intestinal architecture and promote accurate immunostaining. Once mastered, this technique can be used to investigate a wide variety of research questions involving intestinal physiology and cell biology19. Several optimized Swiss rolling methods have been published and are very useful20,21. An advantage of this technique is the ease of accurately opening the intestine on filter paper. This allows tissue to be fixed flat, preventing tissue from curling inwards when rolling, which is especially helpful when analyzing inflamed tissue with thickened muscularis. In addition, it is essential to highlight the critical role of tissue sectioning in achieving reliable results. Proper sectioning ensures the preservation of tissue architecture and facilitates accurate immunostaining, ultimately contributing to the success of downstream analyses11. The optimized approach is less difficult compared to other protocols, yielding consistent results among different individuals. The technique put forth in this paper also provides well-preserved tissue architecture, versatility, and highly reproducible immunostaining using diverse antibodies.
A critical aspect of this protocol is the fixation and processing time. Improper tissue fixation and processing can impair histochemical analyses. Tissue that is over-fixed becomes brittle, while under-fixed tissue stays too soft. Both over- and under-fixed tissues are difficult to section and compromise immunostaining. Once dissected onto filter paper, tissue should be immediately placed in formalin to reduce post-mortem alterations22. In this protocol, murine tissue is fixed in formalin overnight. Several studies have used this fixation time20,23,24. However, Boenisch et al. showed that immunostaining is consistent in tissue fixed in formalin for up to 4 days25. Optimization of fixing time and fixative is required depending on desired analyses. For example, Carnoy’s fixative is often preferred for mucus staining26. The choice of fixatives and duration of fixation time should be optimized by each researcher depending on their experimental approach. The use of an automated tissue processor is recommended to ensure accurate and consistent processing times. Our laboratory uses small pins to hold intestinal tissue in place as rolls. Without pins, tissue may come unrolled during processing. As these pins are quite small and sharp, precautions must be taken. We advise using wire cutters to remove the sharp end of the pin prior to tissue processing. Some histology cores will not accept tissue with pins; therefore, it is best to check before using. An alternative approach is to use agar to anchor tissue before processing27 or cassette sponges could be used in cassettes to help maintain rolls.
Immunostaining is a protocol that requires optimization for each antibody. The use of knockout-validated antibodies is recommended whenever possible. This method for tissue fixation and processing results in clear staining, allowing for easy identification of antibody-binding compared to background signal when non-validated antibodies are being utilized. Fixative choice, antigen retrieval, and antibody dilution can impact the specificity of the antibody. We recommend that researchers carefully assess and optimize the protocol by adjusting fixation, antigen retrieval, and incubation time for each antibody. To obtain the best results, antibodies should be tested at various dilutions to determine optimal concentration and in different antigen retrieval buffers. Epitope retrieval improves immunostaining by breaking methylene bridges formed during fixation. In this protocol, heat-induced epitope retrieval is used rather than proteolytic-induced epitope retrieval because enzyme digestion is more likely to disrupt tissue morphology28. The most common heat-induced epitope retrieval buffers are citrate buffer, tris-HCl, and tris-EDTA, with citrate buffer being the gentlest on tissue morphology29. Buffer choice varies and should be determined for each antibody. Many antigen retrieval buffers, blocking solutions, and antibody diluents are available commercially. However, these solutions can be extremely expensive and cost-prohibitive. We provided recipes of common antigen retrieval solutions and blocking and antibody diluent solutions to ensure cost-effectiveness.
A limitation of this method is that fixation and processing can alter tissue and mask epitopes. An alternative approach is to immunostain fresh frozen tissue. Fresh frozen tissue is snap frozen, allowing avoidance of exposure to toxic fixatives, preserving protein structure, and improving accessibility of some epitopes. However, tissue architecture and morphology are poorer than those of fixed and paraffin-embedded tissue. Further challenges of fresh frozen tissue include the materials and logistics required to section and store frozen blocks and slides. Analysis of Swiss rolls versus strips of intestinal tissue shows differences in villi height and width and differences in immune cells in the lamina propria, as described in an earlier report30. These results suggest that intestinal Swiss rolls alter some intestinal features, which should be considered when planning experiments. Additionally, antibody revalidation is required as many antibodies that bind specifically to formalin-fixed paraffin-embedded tissue do not stain well in fresh frozen tissue31.
Immunostaining of intestinal tissue can be used to answer a wide variety of questions regarding gastrointestinal cell biology and physiology. This technique is widely used to identify epithelial alterations in the setting of intestinal inflammation, following bacterial infection, and during cancer progression. The method presented here is ideal for the preservation of intestinal tissue because it is cost-effective, not technically challenging, and highly reproducible. To ensure the best results, we encourage optimization of the steps outlined in this protocol based on the experimental design and the hypothesis being tested.
The authors have nothing to disclose.
This study was supported by the National Institutes of Health (NIH) grants K01 DK121869 to ACE and this publication was supported in part by T32 GM132055 (RME), F31 DK139736 (SAD), T32 DK124191 (SAD), TL1 TR001451 (RS), UL1 TR001450 (RS) and the HCS cornerstone grants to SAD & RS. This work was supported by startup funds from the Medical University of South Carolina (MUSC) to ACE and was supported by the MUSC Digestive Disease Research Core Center (P30 DK123704) and the COBRE in Digestive and Liver Disease (P20 GM120475). Imaging was performed using the cell and molecular imaging core at MUSC.
β-CATENIN | GeneTex | GTX101435 | |
Cellulose filter paper | Cytiva | 10427804 | Thick Whatman paper |
Charged glass slides | Thermo Fisher Scientific | 23888114 | |
Coverslip | Epredia | 152440 | |
Dissecting pins size 00 | Phusis | B082DH4TZF | |
E-CADHERIN | R&D Systems | AF748 | |
Freezer gloves | Tempshield | UX-09113-02 | |
Heating block | Premiere | XH-2001 | Slide Warmer |
Histo-Clear II | Electron Microscopy Sciences | 64111-04 | Clearing reagent |
Hoescht | Thermo Fisher Scientific | 62249 | |
Hydrochloric Acid | Sigma Aldrich | 320331 | |
Hydrophobic pen | Millipore | 402176 | |
LAMININ | GeneTex | GTX27463 | |
LAMP1 | Santa Cruz | SC-19992 | |
Large cassettes | Tissue-Tek | 4173 | |
Minutien pins | Fine Science Tools | NC9679721 | |
Mouse-on-mouse blocking reagent | Vector Laboratories | MKB-2213 | Mouse-on-mouse block |
MUC2 | GeneTex | GTX100664 | |
PCNA | Cell Signaling Technology | 2586S | |
Pressure Cooker | Cuisinart | B000MPA044 | |
ProLong gold antifade | Thermo Fisher Scientific | P36934 | Mounting medium |
Reverse action forceps | Dumont | 5748 | |
Slide Rack | Tissue-Tek | 62543-06 | |
Slide Staining Set | Tissue-Tek | 62540-01 | Solvent Resistant Dishes and Metal Frame |
Small cassettes | Fisherbrand | 15-200-403B | |
Sodium citrate dihydrate | Fisher Bioreagents | BP327-1 | |
Teleostein Gelatin | Sigma | G7765 | Blocking buffer |
Triton X-100 | Thermo Fisher Scientific | A16046 | |
Tween 20 | Thermo Fisher Scientific | J20605-AP | |
Wipes | KimTech | 34155 | |
Xylenes | Fisher Chemical | 1330-20-7 | |
γ-ACTIN | Santa Cruz | SC-65638 |
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