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 commonly processed and scored by histopathology core facilities and experienced human pathologists through standardized histopathological criteria and methods. Conversely, murine tissues are usually fixed, embedded and analyzed by researchers with limited experience of histopathological protocols. The quality and reliability of histopathological examination begins with the preparation of high-quality tissue sections. Several factors critically contribute to increase or decrease the quality of the final analysis, including fixation, macroscopic sectioning, processing, paraffin embedding, and embedding of the samples^6,7.

All these passages involving manipulation of the sample are subjected to manual errors, including manual embedding of the samples and, to a lesser extent, manual microtome sectioning and staining. At present, the whole process of murine tissue preparation for histological evaluation relies on protocols that vary from laboratory to laboratory and manual protocols. The goal of this study is to implement standardized automated protocols to reduce errors and variability in murine histopathological examination.

To our knowledge, we describe here the first protocols for fully automated tissue processing and embedding for the histological evaluation of murine tissues; these are routinely used in pathology units for the analyses of human specimens. As a practical example of the feasibility of the method, a murine model of intestinal inflammation has been analyzed, i.e., the chronic colitis model caused by repeated administration of dextran sodium sulphate (DSS) in the drinking water^8,9. This experimental setting closely resembles human inflammatory bowel diseases (IBD)¹⁰ since DSS-treated animals exhibit signs of intestinal inflammation, e.g., weight loss, loose stool or diarrhea, and shortening of the colon as well as fibrosis^8,9,11. As observed for human IBD patients, DSS treatment generates a complex disease course. In this context, elaborate histological evaluations are required to understand the profound alteration of the tissue architecture. Thus, the implementation of the described protocols for increasing sample preparation quality might benefit researchers relying on the interpretation of histological and immunohistochemical analyses for murine experimental settings. Murine experimental models of human diseases involving alterations of the tissue architecture, the presence of cellular tissue infiltrate or inflammation in different tissues and organs (intestine, brain, liver, skin) could use the increased quality of the sample preparation for histopathological examination.

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 inflammation⁹.
3. After 7 days, stop DSS treatment and give water to both groups for 14 days. Repeat this process 3 times.
   NOTE: Repetitive administration of DSS induces alterations of the colonic mucosa closely resembling human IBD, i.e., fibrosis⁹.
4. Sacrifice mice according to the procedures authorized by the Institutional IACUC, i.e., by CO₂ inhalation.
5. Open the mouse abdomen and peritoneum with a scalpel.
   NOTE: Sterility is recommended but not strictly required.
6. Separate the colon from the small intestine with forceps and tweezers. Excise the colon with tweezers and forceps.
   NOTE: Colon length measurement (Figure 1A) is a method to determine if colitis occurred in DSS-treated mice.
7. Rinse the colon in a Petri dish with 10 mL of cold 1x phosphate buffer saline (PBS) and gently press to remove fecal material.

2. Murine Tissues Fixation

1. Immerse each murine colon specimen in 10 mL of 10% Neutral Buffered Formalin (NBF) at RT (room temperature).
   NOTE: Sterility is recommended but not strictly required.
2. Fix the tissue for 18–24 h at RT.

3. Colon Sectioning and Tissue Preparation

1. Remove fixed tissues from the NBF container with small tweezers and put them in a Petri dish. Put the colon on a sectioning work plate with small tweezers.
2. Cut colons in fragments (0.2 cm to 0.3 cm lengths) with a sterile scalpel. This fragment length is optimal in order not to exceed the thickness of the cassette.
3. Pick up one colon segment with small tweezers (Figure 2A). Insert one colon segment into one of the plastic protruding tips of the orientated paraffin embedding cassette with small tweezers (Figure 2B).
4. Repeat the operation (3.2–3.3) with an additional 3 colon segments per cassette. Avoid inserting the segments into adjacent protruding tips, to minimize overlapping of the tissues
5. Tightly close the cassette by carefully pushing the four edges (Figure 2C). Avoid squeezing the tissue to prevent tissue damage.
6. Insert the grid into a plastic supporting frame. Label the supporting frame to identify the sample. Repeat for each biological sample.

4. Tissue Processing

1. Turn on the automated processor by pushing the power button (Figure 3A, 3B).
2. Warm up for 1 h to ensure paraffin wax melting. Wait until the instrument confirms the paraffin is completely melted, by observing the presence of the dedicated icon (Figure 3C).
3. Insert each oriented cassette (containing the tissue specimens) manually into the metal basket provided by the automated processor (Figure 3D). Place the cassettes vertically by lining them close to one to the other to optimize the basket occupancy.
4. Close the metal basket (Figure 3E).
5. Open the lid of the retort (Figure 3F). The retort is the place where the basket is inserted into the machine. Insert the basket into the dedicated housing of the processor (Figure 3G). Close the lid of the retort (Figure 3H).
6. Use the touch screen on the instrument computer to define the working protocol (Figure 3I). Choose the sequence of solutions, timing and temperature to be implemented according to the scheme provided in Table 1.
7. Assign the protocol to be run on the retort containing the basket by clicking on the dedicated computer icon (Figure 3J). Start the protocol by clicking on the Start Button (Figure 3L).
8. Wait until the instrument confirms the end of the protocol, by observing the presence of the dedicated icon and by hearing the alarm tone coming from the machine.
9. Open the retort lid. Remove the basket from the processor (Figure 3M).

5.  Tissue Embedding

1. Turn on the automated embedder by pushing the power button (Figure 4A).
2. Warm up for 1 h to ensure paraffin wax melting. Wait until the instrument confirms the paraffin is completely melted by observing the temperature of the paraffin bath indicated by the internal thermometer of the instrument.
3. Manually transfer all the processed cassettes from the processor basket to the embedder rack. Each rack can contain up to 32 cassettes. (Figure 4B, 4C).
4. Open the main embedder lid (Figure 4C, 4D, 4E).
5. Use the touch screen on the instrument computer to signal to the robotic system that a rack is being inserted (Figure 4F).
6. Open the inlet housing lid (Figure 4G). Insert the rack into the inlet housing. Each embedder can contain up to 4 racks simultaneously (Figure 4H, 4I). Close the inlet housing lid (Figure 4J).
7. Use the touch screen on the instrument computer to start the embedding procedure, according to Table 2 (Figure 4K).
   NOTE: Each rack of 32 cassettes takes 45 min to be embedded.
8. Wait until the instrument confirms the end of the protocol, by observing the presence of the dedicated icon (Figure 4L).
9. Remove the outlet rack (Figure 4M). Close the main embedder lid.
10. Remove the embedded blocks (containing the orientation grids) from the rack. Transfer the embedded blocks in a storage cardboard box.

6. Micrometer Sectioning

1. Turn on the cooling plate of the microtome. Set the temperature between -8 and -10 °C.
2. Turn on the thermostatic water bath of the microtome, containing 2 L of distilled water. Set the temperature of the water bath between 42–45 °C.
3. Place the paraffin embedded blocks on the cooling plate. Wait at least 5 min to allow the blocks to cool.
4. Take one block from the cooling plate and place it into the microtome block holder.
5. Set the microtome cut thickness to 10 µm. Trim the block by cutting it 6 times at 10 µm thickness.
6. Change the thickness setting of the microtome from 10 µm to 3 µm. Cut one 3 µm section for Hematoxylin and Eosin (H&E) staining.
7. Collect the 3 µm section with a small brush. Put the 3 µm section in the water bath, laying it carefully on the water surface to reduce tissue wrinkles. Collect the tissue section from the thermostatically controlled water bath on a single glass slide.
   1. If needed, cut additional sections.
8. Label the glass slide by writing the sample identification.
9. Put the slides in a 37 °C oven for at least 10 min.
10. Put glass slides into racks for immediate analyses or in storage boxes.

7. Hematoxylin and Eosin (H&E) Staining

1. Switch on the automated stainer by pushing the power button. Allow the initialization of the robotic arm, by observing the change of the loading bar on the instrument monitor.
2. Insert the glass slides into the stainer rack, vertically inserting up to 30 slides.
3. Label the rack with the appropriate radio frequency identification (RFID) plastic tag. RFID tagging is a system identifying the correct staining protocol loaded into the stainer computer.
4. Insert the rack into the stainer. Allow the computer to automatically load and start the staining protocol according to the recognition of the RFID tag.
   NOTE: The protocol for H&E staining is described in Table 3.

8. Immunohistochemical Staining

1. Perform immunohistochemical and Mallory trichrome staining as previously described⁷.

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