This work was supported by Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP 2010/19547-1; 2018/03677-5) to MMAB, by Funda&#231;&#227;o de Apoio ao Ensino, Pesquisa e Assist&#234;ncia- FAEPA to MMAB and by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior- Brasil (CAPES) - finance code 001. CG-C received a master and doctoral fellowship from CAPES and LAMT-S received doctoral fellowship from CNPq. We thank Elizabete R. Milani for confocal microscopy assistance and Dr. Dario Zamboni for providing LLC-MK2 cells (Ribeirao Preto Medical School, USP).

1. Cell and parasite cultures
<ol>
	<li>Grow LLC-MK2 (Rhesus Monkey Kidney Epithelial) cells from the American Type Culture Collection (CCL-7) in a 25 cm2&#160;cell culture flask containing in RPMI medium supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum) and antibiotics (100 U/mL Penicillin and 100 &#181;g/mL Streptomycin) at 37 &#176;C in 5% CO2 17.</li>
	<li>Infect LLC-MK2 cells with Trypanosoma cruzi (Y strain) according to a previous protocol 18.</li>
	<li>Collect the supernatant of the LLC-MK2 infected cells (5 mL) in a 15 mL cell culture conical centrifuge tube and centrifuge at 500 x g for 10 minutes and 22 &#176;C to lower cell debris. Keep the tube for 10 minutes at 37 &#176;C to allow trypomastigotes to swim to the supernatant.</li>
	<li>Collect the supernatant in a new conical tube and centrifuge at 2500 x g for 15 minutes at 22 &#176;C. Discard the supernatant and resuspend the pellet containing the parasites in complete RPMI medium to determine cell density by counting cells in a Neubauer chamber.</li>
</ol>
2. Control immunofluorescence protocol
NOTE: Once fixed, it is possible to store plates containing coverslips at 4 &#176;C in 1x PBS (pH 7.2) for one week. To be stored, it is important that the cells have not gone through the permeabilization step.
<ol>
	<li>Settle LLC-MK2 cells (2 x 104) in 24 well plates containing UV sterilized rounded coverslips in RPMI media for 16 hours.</li>
	<li>For infected cells, add a supernatant containing T. cruzi (item 1.4) to each well in proportion (MOI 10:1) and leave for 6 h of infection. Wash coverslips containing infected and non-infected cells five times with PBS solution and fixed with 2% paraformaldehyde in 1x PBS (pH 7.2) for 10 min at room temperature (RT).</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS (pH 7.2).</li>
	<li>Permeabilize the coverslips with 0.2% IGEPAL CA-630 in 1x PBS (PH 7.2) for 10 min at RT.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Incubate coverslips for 30 min at RT with the blocking solution (2% BSA in 1x PBS, pH 7.2).</li>
	<li>Incubate coverslips for 30 min at RT either with mouse monoclonal anti-hnRNPA1 (dilution 1:200) or with mouse polyclonal anti-TcFAZ (dilution 1:100) antibodies diluted in blocking solution.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Incubate the coverslips for 30 min at RT with goat anti-mouse IgG F (ab&#39;)2 (H+L) conjugated to Alexa Fluor 488 (1:600) together with phalloidin conjugated to Alexa 594 (1:300) to stain actin filaments (F-actin) in the host cell diluted in the blocking solution.</li>
	<li>Wash three times with 1x PBS (pH 7.2) for 5 min each.</li>
	<li>Apply a small amount of ProLong Gold antifade mounting reagent with DAPI medium to the surface of the slide.</li>
	<li>Using forceps, gently tilt the coverslip in the mounting medium to prevent bubbles from forming. Once dry, seal the coverslip if desired.</li>
</ol>
3. Double labeling immunofluorescence protocol using monoclonal and polyclonal antibodies raised in the same host
<ol>
	<li>Repeat steps 2.1 to 2.6 described above.</li>
	<li>Incubate coverslips containing infected and non-infected cells with in-house mouse polyclonal anti-TcFAZ antibody (1:100) diluted in blocking solution for 30 min at RT.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Incubate coverslips for 30 min at RT with goat anti-mouse IgG F (ab&#39;)2 (H+L) conjugated to Alexa Fluor 647 (1:600) diluted in the blocking solution.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Perform the second blocking step with AffiniPure rabbit anti-mouse IgG (H+L) diluted (0.12 mg/mL) in blocking solution for 30 min at RT.</li>
	<li>Wash the coverslips three times with 1x PBS (pH 7.2) for 5 min each. Then incubate with mouse monoclonal anti-hnRNP A1 IgG2b antibody (1:200) for 30 min at RT.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Incubate the coverslips for 30 min with goat anti-mouse IgG2b antibody conjugated to Alexa Fluor 488 (1:600) and with phalloidin conjugated to Alexa 594 (1:300) diluted in the blocking solution.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Repeat steps 2.8 to 2.10 described above.</li>
</ol>
4. Triple labeling immunofluorescence protocol with the addition of the third polyclonal antibody from different host species
NOTE: For the additional labeling, note the IgG subclasses, antibody isotypes, and follow the order of antibodies: 1. mouse polyclonal, 2. rabbit polyclonal, 3. second block, and 4. mouse monoclonal. Consider the type of lasers available in the confocal microscope to choose the correct fluorophore-conjugated secondary antibody.
<ol>
	<li>Repeat steps 2.1 to 2.6 described above.</li>
	<li>After steps 3.2 to 3.5 (washing step), start a new incubation with rabbit polyclonal antibody in blocking solution for 30 minutes at RT.</li>
	<li>Wash the coverslips three times with 1x PBS (pH 7.2) for 5 minutes each.</li>
	<li>Incubate the coverslips with goat anti-rabbit antibody IgG conjugated to Alexa 647 (1:600) in blocking solution for 30 minutes.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Perform a blocking step with AffiniPure rabbit anti-mouse IgG (H+L) diluted (0.12 mg/mL) in blocking solution for 30 min at RT.</li>
	<li>Wash the coverslips three times with 1x PBS (pH 7.2) for 5 minutes each and incubate with any mouse monoclonal IgG subclass antibody diluted in blocking solution for 30 minutes.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
	<li>Incubate the coverslips for 30 minutes with a specific pair of goat anti-mouse IgG subclass antibody conjugated to Alexa 594 (1:600) in blocking solution for 30 minutes.</li>
	<li>Wash the coverslips three times for 5 minutes each with 1x PBS.</li>
</ol>
5. Confocal imaging acquisition
<ol>
	<li>Analyze the immunofluorescence samples using a confocal microscope, with a 63X oil immersion objective and detect the fluorescence with a photomultiplier tube (PMT) and Hybrid detector (HyD). 
	NOTE: We used the setup from the Multiuser Laboratory of Confocal Microscopy - LMMC, Ribeirao Preto Medical School, University of Sao Paulo.</li>
	<li>Acquire all confocal images with separated channels. Perform image processing using Adobe Photoshop.</li>
</ol>

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The authors declare no competing or financial interests.

Here, we present a protocol to perform double immunolabeling in Trypanosoma cruzi infected cells using two different antibodies from the same host species. To study, with more detail, the implications of the infection, structures in the host cell such as the nucleus or cytosolic organelles can be labeled using this protocol. Also, it can be used in the post-embedding thin section immunogold labeling method. This approach helps to overcome the obstacle of having few antibodies available to study trypanosomatids and other parasites.
Additionally, our protocol showed an intracellular parasite is labeled together with the host cell, two eukaryotes in the same immunofluorescence, different from those protocols realized in different kinds of tissues7,8,9,10,11,12,13,14. Our protocol shows that no epitope of the primary polyclonal antibody can be recognized by the second secondary antibody (step 3.9), ensuring that no cross-reactions occur between the antibodies (Figure 3). The success of this methodology is due to the efficiency of the blocking step (step 3.6) using purified rabbit anti-mouse IgG. A similar protocol described by Ansorg et al. (2015) has two additional blocking, where the first uses serum from the same host as the primary antibody and the second is realized with unconjugated Fab-fragments6. Also, without the second blocking, false-positive labeling can occur, as explained by these authors 6.
Furthermore, for triple immunolabeling, the third antibody of a rabbit host species can be added before the second blocking step described above in the protocol (step 4.2). Also, it is possible to use more than one monoclonal antibody since they have different IgG subclasses. For this, the corresponding secondary antibodies should be highly adsorbed to minimize cross-reactivity between them. The use of different IgG subclasses allows the utilization of the same host antibodies without cross-reaction. These protocols work well, but control groups are necessary to avoid false-positive results. The specificity of IgG subclasses and their properties makes it possible to do double and triple labeling. It has been reported that the IgG subclasses differ in the complement activation and Fc receptors in the inter-heavy chain disulfide bonds, hinge region amino acids, molecular mass (kDa), and the relative abundance (in percentage) in response to proteins, saccharides, and allergens 25. They have slight differences in the hinge structure amino acids, influencing the stability and flexibility of each IgG subclass. IgG2 has the shortest and even more rigid hinge of all IgG subclasses due to a CH2 region with one amino acid deletion and an extra inter-heavy chain disulfide bridge 25.
In summary, the protocol described here to study host-pathogen interactions presents a basic and elaborated technique adapted to any combination of antibodies. This approach makes immunofluorescence cost-effective and can help when the source of antibodies is limited. The protocol can simultaneously detect pathogens and the host cell proteins in infected host cells; it can also be applied to any cell type and free-living organisms.

To study the interaction of the pathogen with the host cell at the cellular level provides essential information on the underlying causes of the disease since different groups,&#160;such as viruses, bacteria, and protozoa,&#160;can infect most host cell types1,2,3,4. It can also help develop and identify potential therapeutic targets that can slow or inhibit the growth of the pathogen. In live conditions, the produced antibodies are responsible for recognizing self-components, antigens from viruses, bacterial components or products, fungi, parasites, and others5.
For this purpose, antibodies are widely used tools, mainly for understanding the location and function of cellular structures and proteins. Several studies using multiple antibody labeling demonstrate that additional blocking steps contribute to the specificity of the immunolocalization. In addition, most described protocols use specific commercial monoclonal antibodies, including antibodies from the same host species6,7,8,9,10,11,12,13,14.
Usually, double labeling immunofluorescence uses two antibodies raised in different species to stain the cell structures of interest or the pathogens and the host cells to see the interaction between them. However, this can be a problem when no commercial monoclonal or polyclonal antibodies specific for some pathogens are available to perform the double labeling. Also, there are commercially available antibody conjugation kits, and it is possible to conjugate the primary antibodies directly to the fluorophore by a succinimidyl ester reaction15. The problem is that these kits are often expensive, and it is necessary to have enough antibodies to label them. Knowing this, we successfully developed a double immunofluorescence method using two different antibodies raised in the same species to study protein localization in Trypanosoma brucei16. However, for intracellular parasites, including Trypanosoma cruzi, this approach has not been demonstrated. Here, we show how to perform double labeling immunofluorescence to study intracellular T. cruzi parasites and the host cell using primary antibodies raised in the same species without cross-reactions. Besides this method, a triple immunofluorescence labeling has been established with the addition of the third antibody from a different species. These approaches help when the source of antibodies is limited and can be used in any cell type.

<table><tbody><tr><td>Alexa Fluor 488 - IgG2b antibody</td><td>Life technologies, USA</td><td>A21141</td><td>Goat anti-mouse</td></tr><tr><td>AffiniPure Rabbit anti-mouse IgG (H+L)</td><td>Jackson Immunoresearch, USA</td><td>315-005-003</td><td>Anti-mouse antibody</td></tr><tr><td>Alexa Fluor 488 - IgG F (ab&#039;)2 (H+L) antibody</td><td>Life technologies, USA</td><td>A11017</td><td>Goat anti mouse</td></tr><tr><td>Alexa Fluor 594 IgG1 antibody</td><td>Life technologies, USA</td><td>A21125</td><td>Goat anti-mouse</td></tr><tr><td>Alexa Fluor 647 - IgG F (ab&#039;)2 (H+L) antibody</td><td>Life technologies, USA</td><td>A21237</td><td>Goat anti-mouse</td></tr><tr><td>Anti-hnRNPA1 antibody IgG2b</td><td>Sigma-Aldrich, USA</td><td>R4528</td><td>Mouse antibody</td></tr><tr><td>anti-TcFAZ (T. cruzi FAZ protein) antibody</td><td>Our lab</td><td>In-house</td><td>Mouse antibody</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich, USA</td><td>A2153-10G</td><td>Albumin protein</td></tr><tr><td>Detergent Igepal CA-630</td><td>Sigma-Aldrich, USA</td><td>I3021</td><td>Nonionic Detergent</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco, Thermo fisher scientific, USA</td><td>12657-029</td><td>Serum</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco, Thermo fisher scientific, USA</td><td>15140-122</td><td>Antibiotic</td></tr><tr><td>Phalloidin Alexa Fluor 594</td><td>Life technologies, USA</td><td>A12381</td><td>Actin marker</td></tr><tr><td>ProLong Gold antifade with DAPI</td><td>Life technologies, USA</td><td>P36935</td><td>Mounting media reagent</td></tr><tr><td>RPMI 1640 1X with L-glutamine</td><td>Corning, USA</td><td>10-040-CV</td><td>Cell culture media</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich, USA</td><td>T4049-100ML</td><td>Bioreagent</td></tr></tbody></table>

double labeling immunofluorescence using antibodies from the same species to study host-pathogen interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, we show how to study host-parasite interactions by immunofluorescence when the source of antibodies is limited due to the unavailability of commercial antibodies that recognize specific structures and proteins in trypanosomatids.
Among trypanosomatids, T. cruzi has one of the most complex life cycles involving various development stages between vertebrate and invertebrate hosts 19. During the T. cruzi life cycle, at an early stage of mammalian infection, metacyclic trypomastigotes invade cells through a process that involves a wide variety of molecules present in parasites and the vertebrate host cells 19,20. To study these processes, our laboratory routinely produces in-house polyclonal antibodies against proteins of T. cruzi, T. brucei, and Leishmania sp16,21,22,23&#160;to use together with commercial mouse monoclonal antibodies and/or with rabbit antibodies.
In Figure 1, confocal microscopy images show the results with the control experiments of infected and non-infected cells, highlighting the specificity of the antibodies in the host cell and the internalized parasite. The mouse polyclonal antibody (anti-TcFAZ) raised in our lab recognized only T. cruzi giant protein in the FAZ at the parasite flagellum but not in the host cell (Figure 1A). The distribution of the heterogeneous nuclear ribonucleoprotein A124&#160;in the nuclei was observed using a commercial mouse monoclonal anti-hnRNP A1 antibody that recognizes only the host mammalian cells but not the parasite (Figure 1B). Also, host and parasite nuclei and the parasite kinetoplasts were stained with DAPI, and host F-actin was stained with Phalloidin conjugated to Alexa 594 (Figure 1). These control results show the specificity of the antibodies, and then, we can run the double labeling immunofluorescence protocol (Figure 2).
In Figure 2, double labeling immunofluorescence shows the protein distributions in the host and the parasite analyzed by confocal microscopy. No cross-reactions occur between antibodies using this methodology. The efficiency of the second blocking step using purified rabbit anti-mouse IgG is enough to impair the nonspecific labeling by the secondary antibodies. This labeling allows studying the interaction between the parasite and the host proteins and their behavior during infection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62219/62219fig01.jpg" /> 
Figure 1: Confocal Microscopy showing the localization of the T.&#160;cruzi giant protein (TcFAZ) and host hnRNP A1 in non-infected (NI) and infected (INF) LLC-MK2 cells with T. cruzi. (A) T. cruzi giant protein (TcFAZ) localized in T. cruzi flagellum is labeled with mouse polyclonal anti-TcFAZ antibody and visualized with the goat anti-mouse IgG secondary antibody conjugated to Alexa 488 (green). (B) Host nuclear hnRNP A1 in LLC-MK2 is labeled with mouse monoclonal anti-hnRNP A1 IgG2b antibody and visualized by goat anti-mouse IgG2b antibody conjugated to Alexa 488 (green). Phalloidin conjugated to Alexa 594 stains F-actin (red). Nuclei and kinetoplasts are stained with DAPI (blue). Scale bar = 5 &#181;m. All experiments were made in biological triplicate. White arrows indicate intracellular parasites. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62219/62219fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62219/62219fig2v2.jpg" /> 
Figure 2: Double labeling immunofluorescence shows no-cross reaction between antibodies raised in the same host species in non-infected (NI) and infected (INF) LLC-MK2 cells with T. cruzi analyzed by Confocal Microscopy. Giant protein localized in the flagellum attachment zone (FAZ) is labeled with mouse polyclonal anti-TcFAZ antibody and visualized by goat anti-mouse IgG antibody conjugated to Alexa 647 (red). Host nuclear hnRNP A1 is labeled with mouse monoclonal anti-hnRNP A1 IgG2b antibody and visualized by goat anti-mouse IgG2b antibody conjugated to Alexa 488 (green). Phalloidin conjugated to Alexa 594 stains F-actin (grey). Nuclei and kinetoplasts are stained with DAPI (blue). Merged images are shown as indicated. Inset corresponds to the enlarged area of the host nucleus. Bar=5 &#181;m. All experiments were made in biological triplicate. The white arrow shows the presence of the parasite near the host cell nucleus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62219/62219fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62219/62219fig03.jpg" /> 
Figure 3: Schematic illustration of&#160;sequential double immunostaining with primary antibodies derived from the same species. Diagram shows the order of the antibodies to ensure the efficiency of this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62219/62219fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions at JoVE.com

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

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Immunology and Infection

6.3K Views.  University of Sao Paulo, Ribeirao Preto, SP, Brazil. In dit protocol kunnen twee antilichamen van dezelfde gastheer samen worden gebruikt in de immunofluorescentietest om gastheercel- en pathogeeninteracties te bestuderen. Omdat er slechts enkele commerciële antilichamen beschikbaar zijn om specifieke celstructuren en eiwitten in parasieten te herkennen, kan dit protocol zeer nuttig zijn. Dit is een eenvoudig uit te voeren dubbel labelend immunofluorescentieprotocol dat polyklonale en monoklonale antilichamen gebruikt die in dezelfde soort zij...

Video: Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Neuroscience

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals &#8722; Using Unconjugated Primary Antibodies from the Same Host Species

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.

Several different methods have been established for multiplex immunostaining using primary antibodies from the same host species. Here, we describe the use of microwave-mediated antibody stripping and of fluorophore-tyramide to block antibody cross-reactivity during multiplex immunostaining on formalin-fixed paraffin-embedded mouse adrenal sections.

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling ...

Developmental Biology

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Biology

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

Medicine

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201 
2 Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Confocal fluorescence microscopy is an imaging technique that enables increased optical resolution as compared to conventional &#34;wide-field&#34; epifluorescence microscopy. Confocal microscopes are able to achieve improved x-y optical resolution through &#34;laser scanning&#34;- typically a set of voltage-controlled mirrors (galvanometer or &#34;galvo&#34; mirrors) that direct laser-illumination to each pixel of the specimen at a time. More importantly, confocal microscopes achieve superior z-axial resolution by using a pinhole to remove out of focus light originating from locations that are not in the z-plane being scanned, thus enabling the detector to collect data from a specified z-plane. Because of the high z-resolution achievable in confocal microscopy, it is possible to collect images from a series of z-planes (also-called z-stack) and construct a 3D image through software.
Before discussing the mechanism of a confocal microscope, it is important to consider how a sample interacts with light. Light is composed of photons, packets of electromagnetic energy. A photon impinging on a biological sample can interact with the molecules comprising the sample in one of four ways: 1) the photon does not interact and passes through the sample; 2) the photon is reflected/scattered; 3) the photon is absorbed by a molecule and the absorbed energy is released as heat through processes collectively known as nonradiative decay; and 4) the photon is absorbed and the energy is then rapidly reemitted as a secondary photon through the process known as fluorescence. A molecule whose structure permits fluorescence emission is called a fluorophore. Most biological samples contain negligible endogenous fluorophores; therefore exogenous fluorophores must be used to highlight features of interest in the sample. During fluorescence microscopy, the sample is illuminated with light of the appropriate wavelength for absorption by the fluorophore. Upon absorbing a photon, a fluorophore is said to be &#34;excited&#34; and the process of absorption is referred to as &#34;excitation&#34;. When a fluorophore gives up energy in the form of a photon, the process is known as &#34;emission&#34;, and the emitted photon is called fluorescence.
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a &#34;focal point&#34; where it is maximally focused. Beyond the focal point the light again diverges. The entering and exiting beams may be visualized as a pair of cones touching at the focal point (see Figure 1, left panel). The phenomenon of diffraction imposes a limit on how tightly a beam of light can be focused - the beam actually focuses to a spot of finite size. Two factors determine the size of the focal spot: 1) the wavelength of the light, and 2) the light-gathering ability of the objective lens, which is characterized by its numerical aperture (NA). The focal &#34;spot&#34; extends not only in the x-y plane, but also in the z direction, and is in reality a focal volume. The dimensions of this focal volume define the maximum resolution achievable by optical imaging. Although the number of photons is greatest within the focal volume, the conical light paths above and below the focus also contain a lower density of photons. Any fluorophore in the light path can thus be excited. In conventional (wide-field) epifluorescence microscopy, emission from fluorophores above and below the focal plane contribute out-of-focus fluorescence (a &#34;hazy background&#34;), which reduces image resolution and contrast, as demonstrated in Figure 1, with the red cube representing fluorophore emission above the focal plane (red star) that results in out-of-focus fluorescence (top right). This problem is ameliorated in confocal microscopy, due to the utilization of a pinhole. (Figure 2, bottom right). As depicted in Figure 3, the pinhole allows emissions originating from the focal place to reach the detector (left), while blocking the out-of-focus fluorescence (right) from reaching the detector, thus improving both resolution and contrast.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig1.jpg" /> 
Figure 1. Optical resolution of epifluorescence versus confocal microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10501/10501fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The light beam used to excite a fluorophore is focused by the objective lens of a microscope and converges at a focal volume and then diverges (left). The red star represents the focal plane of a sample that is being imaged while the red square represents fluorophore emission above the focal plane. When capturing an image of this sample using an epifluorescent microscope, the emission from the out-of-focus red square will visible and contribute to a &#34;hazy background&#34; (top right). Confocal microscopes have a pinhole which prevents the detection of light emitted outside of the focal plane, eliminating the &#34;hazy background&#34; (bottom right).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig2.jpg" /> 
Figure 2. Pinhole effect in confocal microscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10501/10501fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the highest intensity of the excitation light is at the focal point of the lens (left, red oval), other parts of the sample not in the focal point (right, red star) will get light and fluoresce. To prevent light emitted from these out-of-focus regions to reach the detector, a screen with a pinhole is present in front of the detector. Only the in-focus light (left) emitting from the focal plane is able to travel through the pinhole and reach the detector. The out-of-focus light (right) is blocked with the pinhole and fails to reach the detector.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10501/10501fig3.jpg" /> 
Figure 3. Principal components of a confocal laser scanning microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10501/10501fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the sake of simplicity, the mechanistic description of a confocal microscope will be limited to that of the Nikon Eclipse Ti A1R. Although there may be minor technical differences between different confocal microscopes, the A1R serves well as a good model for describing confocal microscope function. The excitation light beam, produced by an array of diode lasers, is reflected by the primary dichroic mirror into the objective, which focuses the light onto the specimen being imaged. The primary dichroic mirror selectively reflects the excitation light while allowing light at other wavelengths to pass through. The light then encounters the scanning mirrors that sweep the light beam across the specimen in an x-y manner, illuminating a single (x,y) pixel at a time. Fluorescence emitted by fluorophores at the illuminated pixel is collected by the objective lens and passes through the primary dichroic mirror to reach an array of photomultiplier tubes (PMTs). Secondary dichroic mirrors direct the emission light to the appropriate PMT. Excitation light scattered by the sample back into the objective is reflected by the primary dichroic mirror back towards the specimen, and thus prevented from entering the detection light path and reaching the PMTs (see Figure 3). This allows the relatively feeble fluorescence to be quantified without contamination by light scattered from the excitation light beam, which is typically orders of magnitude more intense than the fluorescence. Because the pinhole blocks light from outside of the focal volume, the light arriving at the detector comes from a narrow, selected z-plane. Therefore, images can be collected from a series of adjacent z-planes; this series of images is often referred to as a &#39;z-stack&#39;. By using the appropriate software, a z-stack can be processed to generate a 3D image of the specimen. A particular advantage of confocal microscopy is the ability to distinguish the subcellular localization of staining. For example, the differentiation between membrane staining from intracellular staining, which is very challenging with conventional epifluorescence microscopy (1, 2, 3).
Sample preparation is an important facet of confocal imaging. A strength of optical microscopy techniques is the flexibility to image live or fixed cells. When attempting to produce 3D images, because of the number of images that must be acquired for a z-stack, the difficulty of maintaining cell health, and the movement of live cells and their organelles, the use of fixed cells is typical. The procedure for fixing and staining cells for confocal fluorescence is similar to that conventionally used in immunofluorescence. After culture in chamber slides or on coverslips, cells are fixed using paraformaldehyde to preserve cellular morphology. Non-specific antibody binding is blocked using bovine serum albumin, milk, or normal serum. In order to maintain the specificity of the secondary antibodies, the solution used should not originate from the same species in which the primary antibodies were generated. The cells are incubated with primary antibodies that bind the antigen of interest. When labeling several cellular targets, the primary antibodies must each be derived from a different species. Antibodies tagging an antigen are then bound by fluorophore-conjugated secondary antibodies. Fluorophore-conjugated secondary antibodies should be selected so that they are compatible with the wavelengths of laser excitation available in the confocal microscope. When visualizing multiple antigens, the excitation/emission spectra of the fluorophores should differ enough so that their signals can be discriminated by microscopic analysis. The stained specimen is then mounted on a slide for imaging. A mounting medium is used to prevent photobleaching and specimen dehydration. If desired, a mounting medium containing a nuclear counterstain (e.g. DAPI or Hoechst) can be used (4).
In the following protocol, mouse fibroblasts transfected to express CD1d (LCD1) were stained with antibodies recognizing CD1d and CD107a (LAMP-1). CD1d is a major histocompatibility complex 1 (MHC 1)-like receptor present on the surface of antigen presenting cells that presents lipid antigens. LAMP-1 (lysosomal associated membrane protein-1) is a transmembrane protein primarily present in lysosomal membranes. For proper antigen presentation, CD1d is trafficked through the low pH lysosomal compartment, so LAMP-1 is being used as a marker of the lysosomal compartment for this protocol. By probing the LCD1 cells with anti-CD1d and anti- LAMP-1 that were produced in different species, secondary antibodies with unique fluorophores can be used to determine the localization of each protein in the cell and whether CD1d is present in the LAMP-1 positive lysosomal compartments.

Confocal Fluorescence Microscopy and Localization of Proteins

Source: Dominique R. Bollino1, Eric A. Legenzov2, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine ...

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A Double-Labeling Immunofluorescence Technique to Visualize Host and Pathogen Proteins in an Infected Cell

Source: Gachet-Castro, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62219">Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2021)
This video showcases double-labeling immunostaining with two different primary antibodies raised in the same species, binding specifically to parasite and host proteins. Furthermore, labeled secondary antibodies targeting these primary antibodies allow the distinct visualization of host and parasite proteins, facilitating the study of host-pathogen interaction.

1. Cell and parasite cultures

	Grow LLC-MK2 (Rhesus Monkey Kidney Epithelial) cells from the American Type Culture Collection (CCL-7) in a 25 ...

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Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Immunofluorescence Microscopy

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4&#8217;,6&#8217;-diamidino-2-phenylindole (DAPI), and acridine orange.
The microscope irradiates the sample with short wavelength excitation, such as ultraviolet or blue light. The chromophores absorb the excitation light and emit visible light of longer wavelengths. The excitation light is then filtered out (partly because ultraviolet light is harmful to the eyes) so that only visible light passes through the ocular lens, producing an image of the specimen in bright colors against a dark background.
Fluorescence microscopes can identify pathogens, find particular species within an environment, or find the locations of particular molecules and structures within a cell. Approaches have also been developed to distinguish living from dead cells based on whether they take up particular fluorochromes. Sometimes, multiple fluorochromes are used on the same specimen to show different structures or features.
One of the most important applications of fluorescence microscopy is immunofluorescence, which is used to identify certain microbes by observing whether antibodies bind to them. (Antibodies are protein molecules the immune system produces that attach to specific pathogens to kill or inhibit them.) This technique has two approaches: direct immunofluorescence assay (DFA) and indirect immunofluorescence assay (IFA). In DFA, specific antibodies (e.g., those that target the rabies virus) are stained with a fluorochrome. If the specimen contains the targeted pathogen, one can observe the antibodies binding to the pathogen under the fluorescent microscope. This is a primary antibody stain because the stained antibodies attach directly to the pathogen.
In IFA, secondary antibodies are stained with a fluorochrome rather than primary antibodies. Secondary antibodies do not attach directly to the organism but bind to primary antibodies. When the unstained primary antibodies bind to the pathogen, the fluorescent secondary antibodies can be observed binding to the primary antibodies. Thus, the secondary antibodies are attached indirectly to the pathogen. Since multiple secondary antibodies can often attach to a primary antibody, IFA increases the number of fluorescent antibodies attached to the specimen, making it easier to visualize its features.
This text is adapted from Openstax, Microbiology 2e, Section 2.4: Staining Microscopic Specimens.

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

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