CLEM with Commercially Available Reagents to Facilitate Immunolocalization
Leslie Cummins,1,5 * Vincent Tu,3
and Vera DesMarais1,2,5 1
Joshua Mayoral,3 Louis M. Weiss,3,4 Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx, NY
2Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, Bronx, NY 3Department of Pathology Albert Einstein College of Medicine, Bronx, NY 4Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 5Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY
*
leslie.gunther@
einsteinmed.org
Abstract: There is often a need to locate the same cellular structure of interest in light and electron microscopy, which can be a diffi- cult task. Here we present a method that uses only commercially available reagents and standard epi-fluorescence and transmission electron microscopy (TEM) technology to make correlative light and electron microscopy (CLEM) available to a large group of research- ers without specialized CLEM hardware. This was achieved by seeding cells on photo-etched gridded cover slips and staining the protein to be localized with a secondary antibody coupled to both a fluorophore and 10 nm gold. The presence of the grid allowed for the alignment of light microscopy images with TEM images and the double-labeled antibody revealed co-localization of the fluorophore with gold particles.
Keywords: transmission electron microscopy, microscopy,
fluorescence Toxoplasma gondii
Introduction In order to examine cellular localization of a protein of
interest with a particular biological structure, it is common to utilize primary antibodies against the protein with subsequent fluorophore conjugated secondary antibody staining and image acquisition with fluorescence microscopy. When the morphol- ogy of cellular structures needs to be investigated at high reso- lution, transmission electron microscopy (TEM) can be used. However, at times there is a need to investigate the same cell or cellular structure with both fluorescence microscopy and TEM to visualize protein localization with high-resolution morphology. Tis can be achieved by combining fluorescence microscopy with TEM by using correlative light and electron microscopy (CLEM). While there are many high-end CLEM techniques available from a variety of different manufacturers that streamline the process, these oſten require the purchase of expensive specialized equipment [1,2]. We developed an approach to CLEM using only commercially available reagents and standard epi-fluorescence and TEM systems. Tis makes CLEM universally available to researchers who have access to regular fluorescence microscopes and TEMs, but not the specialized equipment typically used to facilitate CLEM. To address these issues, we developed a method to prepare and fix samples on photo-etched, gridded cover slips stained first with a primary and then a secondary antibody conjugated to both an Alexa fluorophore and 10 nm gold. Tis allowed observa- tion of identical fields of view in both fluorescence and TEM systems with a single staining procedure.
20 doi:10.1017/S1551929520001078 microscopy, correlative
immunoelectron microscopy,
Te model system to which we applied this technique was
a monolayer of human fibroblasts infected with an Apicom- plexan intracellular parasite Toxoplasma gondii [3]. We were interested in the biology and localization of a T. gondii brady- zoite-specific protein, termed MAG2, that we had identified in proteomic studies of the cyst wall of T. gondii [4]. Toxoplasma gondii, an Apicomplexan parasite, can cause significant dis- ease in both immune competent and immunocompromised humans. Te predilection of this parasite for the central ner- vous system causing necrotizing encephalitis and for the eye causing chorioretinitis constitutes its major threat to patients. Te development of these diseases is a consequence of the tran- sition of bradyzoites, found within tissue cysts (for example, in the CNS within neurons), into actively replicating tachyzoites. Tissue cysts, therefore, serve as a reservoir for the reactivation of toxoplasmosis when the host becomes immuno-compro- mised with conditions such as AIDS or organ transplantation, or immune-compromised due to impaired immune responses to the pathogen when the infection has been acquired in utero. Understanding T. gondii developmental biology and formation of the cyst wall will provide the necessary underpinning for control strategies such as vaccine development, as well as the development of therapeutic agents that could eliminate latency and prevent reactivation toxoplasmosis.
Materials and Methods Bright-field and fluorescence image acquisition Human
fibroblast cells infected with Toxoplasma gondii (Pru strain [1]) were grown on photo-etched cover slips (EMS #72264-23), fixed with 4% buffered paraformaldehyde, and immunola- beled for MAG2 (TgME49_209755) [4] using murine mono- clonal antibody 20C3 [4] as the primary antibody and Alexa 488/10 nm colloidal gold goat anti-mouse IgG (Invitrogen/ Fisher Scientific #A31561) as the secondary antibody. Images were acquired on a DeltaVision Personal Epifluorescence Microscope (Applied Precision–GE Healthcare). One set of images was acquired with a 20× NA 0.3 air objective and another set with a 60× NA 1.42 oil objective, both in phase con- trast and green fluorescence (excitation filter 470/40 and emis- sion filter 525/50). At 60×, fluorescent images were acquired as z-stacks (step size of 0.2 μm) and deconvolved using SoſtWorx (GE Healthcare) soſtware. TEM image acquisition Aſter imaging of Alexa 488 labeled MAG2, the cells were fixed with 2.5% glutaraldehyde
www.microscopy-today.com • 2020 July Frank P. Macaluso,1,2,5
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