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Biology

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Published: July 13th, 2016

DOI:

10.3791/54161

1Department of Medicine, Stony Brook University School of Medicine, 2Department of Physiology & Biophysics, Stony Brook University School of Medicine
* These authors contributed equally

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

The mammalian intestinal epithelium comprises a single layer of columnar cells. In the small intestine, the proliferative cells are confined to the crypts while differentiated cells occupy the villus region. However, because there are no villi in the large bowel, the proliferative cells are localized to the bottom of the crypts and differentiated cells occupy the upper region of the crypts. The intestinal epithelium undergoes rapid replenishment (about 3 - 5 days) that is driven by continuous division of the proliferative cells within the crypts. The proliferative cells of the crypts are not a homogeneous population and are further subdivided into stem cells and trans....

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1. Mice

  1. All studies involving mice were approved by the Stony Brook University Institutional Animal Care and Use Committee (IACUC). The mice were maintained on a 12:12 hr light-dark cycle.
    1. Commercially obtain C57BL/6 mice. Obtain C57BL/6 mice carrying Klf5 alleles flanked by loxP sites (Klf5fl/fl). These mice were previously described21 and graciously provided by Dr. Ryozo Nagai.
  2. Purchase C5.......

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The Swiss rolling technique in combination with immunohistochemical staining allows for comprehensive analysis of small or large intestinal tissue. The example of H&E staining of a large bowel of a C57BL/6 mouse (Figure 1) is an illustration of the feasibility and the effectiveness of this technique. As shown in Figure 1, the image is able to capture all portions of the colon: proximal, middle, and distal. Thus, it allows for comprehensive histologica.......

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The Swiss rolling technique is a powerful method for preparing intestinal tissue for histological and morphological assessment on a large scale. In contrast to the previously described Swiss-rolling technique, which was originally developed for preparation of frozen sections18,19, the procedure presented here allows prompt intestinal tissue preparation and fixation for formalin fixation and paraffin embedding (FFPE). Compared to frozen tissue, FFPE tissue has much longer shelf life and is the preferred type of.......

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We would like to thank Ainara Ruiz de Sabando for providing H&E images. This work was supported by grants from the National Institutes of Health (DK052230, DK093680 and CA172113) awarded to Dr. Vincent W. Yang.

....

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Name Company Catalog Number Comments
Stainless Steel Dissecting Kits VWR 25640-002
Decloaking Chamber Biocare Medical DC2012
Syringe 10ml VWR 89215-218
Swingsette Tissue embedding/processing cassette with lid Simport M515
Superfrost Plus Slides [size: 25x75x1mm] VWR 48311-703
Manual Slide Staining Set Tissue-Tek/Sakura 4451
Staining Dish Green Tissue-Tek/Sakura 4456
Staining Dish White Tissue-Tek/Sakura 4457
24-Slide Slide Holder with Detachable Handle Tissue-Tek/Sakura 4465
Oven Thermo Scientific 6243 for baking slides at 65 degree
Dissection microscope Zeiss Stemi 2000C
Fluorescence Microscope Nikon Eclipse 90i Bright and fluoerescent light, with objectives: 10x, 20x
PAP Pen Super-Liquid Blocker Mini Fisher Scientific DAI-PAP-S-M
Ethanol 200 proof AAPR 111000200
Methanol VWR BDH1135-4LP
Glacial acetic acid AAPR 281000ACS
Xylene Fisher Scientific X5P-1GAL
Hydrogen peroxide 25% solution in water ACROS 202465000
10% bufered formalin Fisher Scientific 22-026-213
Bovine serum fraction V, heat shock Roche 3116956001
Tween 20 Sigma Aldrich P7949
Sodium citrate Fisher Scientific S279
Gavage needle VWR 20068-624
Rabbit anti Klf5 antibody Santa Cruz Biotechnology sc-22797 Dilution 1: 150
Chicken anti EGFP antibody Millipore AB16901 Dilution 1: 500
Rabbit anti Ki67 antibody Biocare Medical CRM325B Dilution 1: 500
Mach3 rabbit AP polymer detection kit Biocare Medical M3R533L
Warp red chromogen kit Biocare Medical WR806 H
Lgr5-EGFP/CreERT2 mice  Jackson labs 008875 
Automated processor Leica Leica TP1020

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