Immunology and Infection
Published: September 27th, 2018
The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It is relevant for testing the intestinal dynamics of oral prophylactics.
Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 µL to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.
The goal of this article is to describe in depth a straightforward method for oral intubation of zebrafish, along with useful associated downstream procedures. Oral intubation using zebrafish has become a practical model in the study of infectious disease dynamics, oral vaccine/immunostimulant, drug/nanoparticle uptake and efficacy, and intestinal mucosal immunity. For example, zebrafish oral intubation has been used in the study of Mycobacterium marinum and Mycobacterium peregrinum infection1. Lovmo et al. also successfully used this model to deliver nanoparticles and M. marinum to the gastro-intestinal trac....
All experimental procedures involving zebrafish (Danio rerio) were authorized by the Ethics Committee of the Universitat Autònoma de Barcelona (CEEH number 1582) in agreement with the International Guiding Principles for Research Involving Animals (EU 2010/63). All experiments with live zebrafish were performed at 26–28 °C.
1. Preparing the Equipment for Oral Intubation
Zebrafish (average weight: 1.03 ± 0.16 g) of mixed sex were successfully intubated with different recombinant protein nanoparticles (bacterial inclusion bodies) using our home-made oral intubation device (Figure 1). We have successfully performed the oral intubation and achieved a low average percentage mortality (6.8%) (Table 1). Zebrafish were either intubated with 30 µL or 50 µL of nanoparticle suspensions and the mortality .......
This protocol is an improvement of the previously described technique for oral intubation by Collymore et al.4 Our protocol describes in detail the oral intubation method and includes the preparation of the intestine for downstream analyses. Our method improves fish manipulation speed allowing one person to perform the whole protocol rapidly, without much variation between operators. A main difference of our protocol with the previous one is that we evaluate the success of an oral intubat.......
This work was supported by grants from the Spanish Ministry of Science, European commission and AGAUR funds to NR (AGL2015-65129-R MINECO/FEDER and 2014SGR-345 AGAUR). RT holds a pre-doctoral scholarship from AGAUR (Spain), JJ was supported by a PhD fellowship from the China Scholarship Council (China) and NR is supported by the Ramón y Cajal program (RYC-2010-06210, 2010, MINECO). We thank Dr. Torrealba for expert advice in protein production, N. Barba from the "Servei de Microscopia" and Dr. M. Costa from the "Servei de Citometria" of the Universitat Autònoma de Barcelona for helpful technical assistance.....
|0.30 mm inner diameter and 0.64 mm outer diameter
|Luer lock needle
|31 G, Kel-F Hub
|Luer lock syringe
|100 μL, Kel-F Hub
|Filtered pipette tip
|Dulbecco's modified eagle medium
|Penicillin and streptomycin
|30 µm mesh size filters with 2 mL reservoir
|Tissue-Tek O.C.T. compound
|Plastic molds for cryosections
|Disposable Vinyl molds. 25 mm x 20 mm x 5 mm
|SuperFrost Plus slide
|Atto-488 NHS ester
|Maxwell RSC simplyRNA Tissue Kit
|Inside of Maxwell RSC simplyRNA Tissue Kit
|adding 20 μl 1-Thioglycerol to 1 ml homogenization solution (2%)
|vertical laboratory rotator
|Suministros Grupo Esper
|5 mL round bottom tube
|Thermo Fisher Scientific
|Agilent 2100 Bioanalyzer System
|iScript cDNA synthesis kit
|CFX384 Real-Time PCR Detection System
|iTaq universal SYBR Green Supermix kit
|Thermo Fisher Scientific
|Fluoroshield with DAPI
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