Efficient Techniques for Comprehensive Sampling of Accessible Tissues in Adult Xenopus

Summary

This is part one of a comprehensive Xenopus sampling protocol. The tissues sampled are the heart ventricle, arterial trunk, left liver lobe, gallbladder, lung, pancreas, spleen, larynx, esophagus, stomach, intestines, testes, fat bodies, oviduct, paired kidneys, sciatic plexus, skin, thymus, and whole eye.

Abstract

Xenopus has long been a pivotal model organism for investigating vertebrate development and disease, offering deep insights into cellular processes and gene function. Despite the wealth of information on embryonic Xenopus, there remains a significant gap in standardized methods for adult tissue sampling, especially for modern approaches like quantitative proteomics. This study introduces a comprehensive protocol for rapid, precise, and efficient sampling of multiple tissues in adult Xenopus. The protocol addresses challenges associated with the subtle anatomical differences compared to other anurans, ensuring reproducibility even for those with limited experience in frog dissection. This protocol is optimized for high-quality biochemical analyses by prioritizing sample freshness. We are facilitating the rapid collection of up to 18 tissues within an hour. Additionally, the methods apply to perfused and unperfused conditions, providing flexibility for a range of experimental needs. This work not only fills a critical methodological gap for Xenopus laevis and tropicalis but also serves as a valuable resource for researchers adapting techniques to similar amphibian models, thereby enhancing the scope and reliability of comparative biological and evolutionary studies.

Introduction

Xenopus offers unique advantages over mice, zebrafish, and other model organisms for studying human disease, particularly in developmental and evolutionary contexts. Its external embryonic development and large, easily manipulable embryos enable real-time observation and experimental interventions (e.g., tissue grafting, gene editing) that are challenging in mice, which develop internally and require invasive procedures. Additionally, Xenopus’ three-chambered heart and tetrapod limbs provide a more direct evolutionary bridge for studying congenital defects¹, limb regeneration², and cardiac develop....

Protocol

All experiments were approved by and performed in accordance with the rules and regulations of the Harvard Medical School IACUC (Institutional Animal Care and Use Committee IS 00001365_3). If perfusion protocol¹³ is followed prior to sampling, skip to step 2.3.

1. Preparation (without perfusion)

1. Ensure that the research institution has approved the euthanasia technique described in this protocol.
2. Prepare a solution of 5 g/L MS-222 (tricaine methanesulfonate) and 5 g/L sodium bicarbonate (see Table of Materials). Check the pH to ensure that it is ≥7.
   NOTE: The volume....

Results

By following all steps of this protocol, the heart ventricle, arterial trunk, left liver lobe, gallbladder, lung, pancreas, spleen, larynx, esophagus, stomach, intestines, testes, fat bodies, oviduct, paired kidneys, sciatic plexus, skin, thymus, and whole eye were cleanly excised within an hour of euthanasia (see Figure 1, Figure 2, Figure 3, Figure 4,

Discussion

The protocol presented here is designed to provide flexibility for users at various experience levels while ensuring the collection of essential tissues in a timely and efficient manner. New users are advised to follow each step in the protocol as outlined to ensure the systematic collection of tissues. More experienced users, however, may opt to skip certain steps and sample only the tissues they require. For instance, accessing the spleen, which is located behind the mesentery, can be achieved without sampling the panc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Corning	21-040-CV
5x Magnifying Glass with LED Light and Stand	amazon.com	B08QJ6J8P1	light must not produce heat
Disposable Transfer Pipets	VWR	414004-036
Dissecting Fine-Pointed Forceps	Fisher Scientific	08-875
Dissecting scissors sharp piont, straight 6.5"	VWR	76457-374
Dissection Tray	Fisher Scientific	14-370-284	styrofoam sheets are an acceptable alternative
Euthanasia container	US Plastic	Item 2860	alternative opaque containers acceptable
Euthanasia container lid	US Plastic	Item 3047
Iridectomy Scissors 6"	VWR	470018-938	iris scissors are an acceptable alternative
MS-222: Syncaine (formerly tricaine)	Pentair AES	TRS1
Rat Tooth Tissue Forceps 5.5 in., Stainless Steel	Fisher Scientific	S08100
Sodium Bicarbonate, Powder, USP	Fisher Scientific	18-606-333
Specimen Forceps, Serrated	VWR	82027-442
T-Pins for Dissecting	Fisher Scinetific	S99385