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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 (Klf5^fl/fl). These mice were previously described²¹ and graciously provided by Dr. Ryozo Nagai.
2. Purchase C5.......

Discussion

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 sections^18,19, 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.......

Materials

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