Published: May 16th, 2023
Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.
Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.
The whole body perfusion of amphibians is routinely completed for the purposes of preservation and fixation1,2,3,4,5,6. However, these procedures occur at a rate that limits the number of fresh samples that can be taken per animal. The goal of this work is to develop an effective blood perfusion protocol in Xenopus, prioritizing the speed of the technique. The protocol takes less than 10 min per animal for X. tropicalis and less than 15 min per X. lae....
All experiments were performed in accordance with the rules and regulations of the Harvard Medical School IACUC (Institutional Animal Care and Use Committee) (IS 00001365_3).
NOTE: Though the primary method of euthanasia described is deemed an acceptable technique for euthanasia by the American Veterinary Medical Association12, it has not been found to lead to the cessation of a heartbeat13. Even the frequently used secondary method of double pit.......
Following successful perfusion, all tissues (excluding the liver in pigmented Xenopus) will be distinctly lighter and less saturated with blood. Major blood vessels will become less noticeable (Figure 10), and tissues (excluding the liver) will rinse cleanly in the buffer after being sampled. While the successful execution of the protocol can ultimately only be confirmed by the quality of the data from exsanguinated tissue samples, several typical problems, their possible causes, an.......
This protocol describes traditional dissection techniques for accessing the coelomic cavity. Other techniques are also acceptable, provided they cause minimal damage to the tissues, the heart is accessible, and the lung and stomach are visible. Similarly, most dissection tools listed can be easily substituted with comparable items.
While attempts have been made to optimize the efficacy of this procedure, results may vary depending on one's experience and variability between individual frogs. O.......
This work was supported by NIH's OD R24 grant OD031956 and NICHD R01 grant HD073104. We thank Darcy Kelly for helpful discussions and initial input on this protocol. We would also like to thank Samantha Jalbert, Jill Ralston, and Wil Ratzan for their assistance and support as well as our three anonymous peer reviewers for their feedback.....
|5x Magnifying glass with LED light and stand
|light must not produce heat
|Disposable transfer pipets
|Dissecting fine-pointed forceps
|Dissecting scissors sharp piont, straight 6.5"
|styrofoam sheets are an acceptable alternative
|alternative opaque containers acceptable
|Euthanasia container lid
|Fine dissection pins
|Living Systems Instrumentation
|General use hypodermic needles, 22 G
|for X. laevis
|General use hypodermic needles, 25 G
|for X. tropicalis
|Heparin, porcine intestinal mucosa
|Iridectomy scissors 6"
|iris scissors are an acceptable alternative
|Luer-to-barb adapter male Luer with lock ring
|size will be dependant on the hosing of the pump used
|Mayo-Hegar needle holder
|mosquito forceps are an acceptable alternative
|MS-222: Syncaine (formerly tricaine)
|Peristaltic liquid pump dosing pump 5–100 mL/min
|any peristaltic pump capable of pumping 5-10mL/min is acceptable
|optional; for dulling needles
|Sodium bicarbonate, powder, USP
|Specimen forceps, serrated
|T-Pins for dissecting
|Ultra-fine short insulin syringes, 31 G
|Wire flush cutters, 6-inch ultra sharp & powerful side cutter clippers
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