Improved Swiss-rolling method for histological analyses of colon tissue

Since the introduction of the Swiss-rolling technique by Reilly and Kirsner in 1965, various methodological approaches have been developed for histological analyses of intestinal tissues. Here, we describe an improved protocol for the processing of freshly harvested murine colons that can be extended to other intestinal tissues. With simple tools, this technique allows to tightly wrap the organ throughout the whole length and to keep it in place before fixation, avoiding excessive stiffness of the tissue. Unlike the original method which relies on frozen samples, processing of the biological samples right after resection preserves epitopes integrity for subsequent immunohistochemical analyses. Ultimately, this method provides a reproducible workflow to capture the entire colon length in a unique histological section in order to assess several features such as intestinal inflammation and tumorigenesis. • Easily include freshly isolated tissues • Shorten preparation time using a few affordable tools • Prevent unrolling and preserve tissue integrity


a b s t r a c t
Since the introduction of the Swiss-rolling technique by Reilly and Kirsner in 1965, various methodological approaches have been developed for histological analyses of intestinal tissues. Here, we describe an improved protocol for the processing of freshly harvested murine colons that can be extended to other intestinal tissues. With simple tools, this technique allows to tightly wrap the organ throughout the whole length and to keep it in place before fixation, avoiding excessive stiffness of the tissue. Unlike the original method which relies on frozen samples, processing of the biological samples right after resection preserves epitopes integrity for subsequent immunohistochemical analyses. Ultimately, this method provides a reproducible workflow to capture the entire colon length in a unique histological section in order to assess several features such as intestinal inflammation and tumorigenesis. a r t i c l e i n f o

Animals
Six-to 8-week-old female C57Bl/6 J mice were purchased from Envigo or bred in the local animal facility. Mice were maintained in the animal facility in specific pathogen-free conditions in a temperature-controlled environment with 12-h light/12-h dark cycles and received food and water ad libitum . Animal experiments followed the Federation of European Laboratory Animal Science Association (FELASA) guidelines and were in compliance with EU Directive 63/2010. Protocol #24,973-2,020,040,413,162,969 v3 was approved by the Charles Darwin Ethical Committee (French Ministry of Research). -Tissue processing/embedding cassette (#M505-2, Simport). -Saffron (natural), flower (#720-0184, VWR).

Method
Swiss rolls [1,2]  Tip: Use the side of the Petri dish as a guide to stay perfectly aligned ( Fig. 1 ) -Fix the roll with a minuten pin using small forceps. The pin has to be placed between the two tines of the fork in order to go through all colon layers.
Tip: Only manipulate minuten pins with small forceps as they are extremely thin (150-μm ∅ ) and easily pierce gloves -Place the Swiss roll in a tube containing 4 mL of 4% PFA at 4 °C for 24 h ( Fig. 1 ). -After fixation, transfer the roll in an inclusion cassette and keep it in 70% EtOH at 4 °C for at least 2 h until paraffin embedding.

Histology
Samples preparation (1) Impregnate swiss rolls using ASP300 tissue processor -Dehydrate tissues with five successive baths of absolute EtOH for 50 min each.
-Clarify tissues in two successive baths of SubX for 30 and 40 min, respectively.
-Imprenate tissues in three baths of paraffin for 35 min each at 60 °C.
(2) Embed samples in paraffin using HistoCore Arcadia H and C -Fill the appropriate mold with liquid paraffin.
-Place the surface of the section at the bottom of the mold to obtain the desired orientation.
-Place the mold on the cold region of Arcadia H to fix the sample.
-Place the cassette over the mold and check that it is within the paraffin.
-Place the cassette and mold on the cold region of Arcadia C.
-Dry at 37 °C until the next day.
-Dewax slides in 2 baths of Bond Dewax solution for 30 s at 72 °C and one bath at room temperature.
-Rehydrate slides in 3 baths containing EtOH 100% each bath for a few seconds.  Representative images of (immuno) histochemical stainings of colon Swiss-rolls. A Hematoxylin, eosin, and saffron (HES) coloration. B CD3 staining revealed with 3,3 -Diaminobenzidine (DAB) with respective zooms on a tertiary lymphoid structure taken with a Plan-Apochromat x20/0.8 objective with a 0.22 μm/pixel numerical resolution.

Slides scan and visualization
-Scan each slide using Zeiss Axio Scan Z1 slide scanner.

Declaration of Competing Interest
JGP is named as inventor on patents for cancer vaccination involving an oncolytic rhabdovirus. These patents have been licensed to Turnstone Biologics of which JGP is shareholder. GK is a cofounder of Samsara Osasuna Therapeutics, everImmune and Therafast Bio. The other authors declare no conflicts of interest.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.