Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

Summary

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding human samples.

Abstract

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Introduction

In the last decades, several experimental models have been developed to dissect the pathogenic mechanisms leading to human diseases^1,2. In order to assess the severity of a disease, researchers must evaluate the effect of a treatment and study the cytological and histological architectural variations or the amount of inflammation³. To perform on those experimental models, detailed histopathological analyses are needed, often comparing murine and human samples^4,5.

Additionally, human samples are common....

Protocol

Animal procedures were approved by the Italian Ministry of Health (Auth. 127/15, 27/13) and followed the animal care guidelines of the European Institute of Oncology IACUC (Institutional Animal Care and Use Committee)

1. Chronic Colitis Induction by Repetitive DSS Administration

1. Separate age and sex matched mice in 2 groups (treatment DSS vs. control H₂O, at least 5 mice littermates per experimental group).
2. Administer 2.5% DSS (40 kDa) in the drinking water for 7 days to the treatment group and water to the control group.
   NOTE: This model induces a chronic transmural intestinal inflam....

Results

Experimental chronic colitis induced by repeated administration of DSS in the drinking water is a murine model of intestinal inflammation closely resembling human IBD^8,9. Figure 1 describes the effects of DSS treatment, including colon shortening (Figure 1A), a widely-used parameter to score the presence of DSS-induced inflammation, and colonic expression of pro-inflammat.......

Discussion

We utilize different automated steps during the preparation of murine tissues for histopathologic analysis. This protocol aims at providing technical hints to increase the reproducibility and the standardization of the whole process, thus enhancing the overall quality of the final histopathological evaluation. We implemented automated instruments and methods for the preparation and embedding of tissues, routinely used in pathology core facilities for the study of human specimens.

To demonstrat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absolute Ethanol anhydrous	Carlo Erba	414605	reagent
Absolute ETOH	Honeywell	02860-1L	reagent
Aluminium Potassium Sulfate	SIGMA	A6435	reagent
Aniline Blue	SIGMA	415049	reagent
carbol Fuchsin	SIGMA	C4165	reagent
CD11b (clone M1/70)	TONBO biosciences	35-0112-U100	antibody
CD20 IHC (clone SA275A11)	Biolegend	150403	antibody
CD3 (17A2)	TONBO biosciences	35-0032-U100	antibody
CD4 (GK1.5)	BD Biosciences	552051	antibody
CD45.2 (clone 104)	BioLegend	109837	antibody
CD8 (53-6.7)	BD Biosciences	553031	antibody
Citrate Buffer pH 6 10x	SIGMA	C9999	reagent
Dab	Vector Laboratories	SK-4100	reagent
DPBS 1x	Microgem	L0615-500	reagent
DSS	TdB Consultancy	DB001	reagent
EDTA	SIGMA	E9884	reagent
EnVision Flex Peroxidase-Blocking Reagent	DAKO		compreso in GV80011-2
EnVision Flex Substrate	DAKO		compreso in GV80011-2
EnVision Flex/HRP	DAKO		compreso in GV80011-2
EnVision Flex+ Rat Linker	DAKO		compreso in GV80011-2
Eosin	VWR	1.09844	reagent
F4/80 (clone BM8)	BioLegend	123108	antibody
Formalin	PanReac	2,529,311,215	reagent
glacial acetic acid	SIGMA	71251	reagent
Goat-anti-Rat-HRP	Agilent DAKO	P0448	antibody
Haematoxylin	DIAPATH	C0303	reagent
LEICA Rotary microtome (RM2255)	Leica	RM2255	equipment
Ly6g (clone 1A8)	BD Biosciences	551459	antibody
Mercury II Oxide	SIGMA	203793	reagent
Omnis Clearify Clearing Agent	DAKO	CACLEGAL	reagent
Omnis EnVision Flex TRS	DAKO	GV80011-2	reagent
Orange G	SIGMA	O3756	reagent
Paraffin	Sakura	7052	reagent
Peloris	LEICA		equipment
Percoll	SIGMA	P4937	reagent
RPMI 1640 without L-Glutamine	Microgem	L0501-500	reagent
STS020	Leica		equipment
Tissue-Teck Paraform Sectionable Cassette	SAKURA	7022	equipment
Tissue-Tek Automated paraffin embedder	Sakura		equipment
Xylene	J.T.Baker	8080.1000	reagent