The authors would like to thank Mr. Ryan Hart and Mr. Arriz Lucas for their invaluable feedback on this communication. This work was supported by NIH NINDS R01 NS065052 and Phoenix Children&#8217;s Hospital Mission Support Funds.

NOTE: All procedures were carried out in compliance with The Institutional Animal Care and Use Committee (IACUC) of the University of Arizona. A list of recommended materials and equipment can be found in Table 1.
1. Tissue Preparation
<ol>
	<li>Perfusion
	<ol>
		<li>Euthanize rodent with an overdose of sodium pentobarbital (25 mg/kg, IP), and perfuse transcardially with phosphate buffered saline (PBS) until completely exsanguinated (3-5 min) at a flow rate of 8 ml/min. For in-depth perfusion instructions, see Gage et al 20129.</li>
		<li>Immediately following PBS flush, fix tissue by perfusing with 4% paraformaldehyde in PBS for 15-20 min at a flow rate of 8 ml/min.</li>
		<li>Remove brain and place in 4% paraformaldehyde for 24 hr, followed by graded sucrose solutions (15%, 30%, 30%, in sequence; prepared in tris buffered saline) at 4&#160;&#176;C. Transfer the brain to the subsequent sucrose solution only after the brain has sunk in each solution. Note: Usually, 5 days in each solution is sufficient time for tissue to sink.</li>
	</ol>
	</li>
	<li>Tissue Freezing and Cryosectioning
	<ol>
		<li>Place the brain in embedding medium, such as O.C.T. compound and submerge in isopentane at a temperature of -35 &#176;C. Allow the brain to freeze for a minimum of 10 min, and then store at -80 &#176;C. Problems can arise if diligence to temperature is not taken; please see discussion for troubleshooting information.</li>
		<li>Cut serial coronal sections at a thickness of 20 &#181;m and a temperature of -20 &#176;C. Collect tissue onto positively charged slides. Brain sections may be placed in a slide box wrapped in foil in a zip-top bag and stored long-term at -80 &#176;C. This method of storage creates a double boundary to prevent exposure to air and frost.</li>
	</ol>
	</li>
</ol>
2. Tissue Processing
NOTE: Example equipment and materials required for staining are shown in Figure 3. Alternatives are available, however, these images will aid those new to immunohistochemical staining to visualize appropriate items prior to purchasing.
<img alt="Figure 3" src="/files/ftp_upload/52293/52293fig3highres.jpg" /> 
Figure 3: Example items required for immunohistochemical staining. The black box shown in (A) is an ideal humidity chamber for immunofluorescence, as slides are protected from light without the need to wrap the box on foil. Following sectioning, slides can be stored in a box such as the yellow box shown in (B). Wrapping the box tightly in foil and placing in a zip-top bag prior to the freezer helps protect the tissue samples from freezer burn. An example of slides is given in (C), with different staining dishes depicted in (D) and (E). Coverslips can vary in size and thickness (F), however 1.2 thick coverslips provide nice imaging results on most upright and confocal microscopes. A pencil such as that shown in (G) can be used to label slides. Permanent markers should be avoided as the ink can run, affecting both staining and the ability to determine what the sample is. A mini pap pen such as that shown in (H) enables a repellent border to be drawn on slides.
<ol>
	<li>Slide preparation
	<ol>
		<li>Remove slides from freezer and thaw at room temperature.
		<ol>
			<li>Optional: If sections have previously floated from slides, place thawed slides in an oven at 60 &#176;C for no more than 4 hrs to help prevent tissue sections from floating off slides.</li>
		</ol>
		</li>
		<li>Place slides in a slide rack and corresponding dish.</li>
		<li>Wash slides three times in PBS for 5 min each, changing solution between washes. From this step forward, avoid having sections without liquid for extended periods of time. Note: If sections dry out, background staining is increased and meaningful data cannot reliably be obtained.</li>
	</ol>
	</li>
	<li>Tissue Staining
	<ol>
		<li>In a light-tight staining box, create a &#8220;humidity chamber&#8221; with lint-free tissues soaked with deionized water.</li>
		<li>Dry the edges of the slide with a lint-free tissue, use a mini pap pen to make a liquid repellent border at the very edge of the slide, away from tissue sections. This border should ensure ample space between the meniscus of the liquid and the edge of the tissue so that surface tension does not affect staining. 
		NOTE: The pap pen repellent border can be applied prior to 2.1.3 if the antibodies of interest do not require microwave antigen retrieval. If the pap pen has been applied prior to washing in PBS, the integrity of the liquid-repellent border must be checked at this step. Use a mini-pap pen to fill in any gaps in the border.</li>
		<li>With slides laid horizontally, block nonspecific antigen binding by incubating in 4% v/v serum in PBS (block solution). Pipette 300 &#181;l of block solution per slide for 1hr at room temperature. Make sure the block solution extends to the pap pen at the edge of the slide and completely covers the tissue to avoid uneven staining caused by surface tension near the tissue.
		<ol>
			<li>Use serum from the same species in which secondary antibody is made. Note: In this procedure, the secondary antibodies are made in donkey, and thus donkey serum is used. If secondary antibodies from two or more different species are used, include serum from each species.</li>
		</ol>
		</li>
		<li>Pipette primary antibody onto slides. Note: Antibody concentrations for this staining have been optimized at 1:5,000 and 1:500 for Iba1 and Pan-neuronal, respectively. These concentrations have been found to show meaningful staining with an absence of background staining.
		<ol>
			<li>Dilute block solution to 1% serum in PBS and add primary antibodies. Pipette 300&#160;&#181;l of primary antibody solution in 1% serum per slide. Again, ensure the fluid is to the edge of the pap pen. Incubate overnight at 4 &#176;C.</li>
			<li>Include three control slides: one that contains neither Iba1 nor Pan-neuronal antibodies, one with Iba1 without Pan-neuronal antibody, and one with Pan-neuronal antibody without Iba1. Stain these slides in the same run with the same solutions, however omit the primary antibodies to test the non-specific binding of the secondary antibodies.</li>
		</ol>
		</li>
		<li>The following morning, wash slides three times in PBS for 5 min each, changing solution between washes.</li>
		<li>Fluorescent antibodies are light-sensitive, therefore, from this step forward, minimize light exposure by ensuring wash containers are wrapped in foil and hybridization boxes are either black or incubated in the dark. Pipette the appropriate secondary antibodies on all slides and incubate for 60 min at room temperature at a concentration of 1:250 in block solution (see step 2.2.3) in a light-tight &#8220;humidity chamber&#8221; (see step 2.2.1).
		<ol>
			<li>Use secondary antibodies of different wavelengths. Here, for the primary antibody rabbit anti-Iba1, use donkey anti- rabbit 594 as the appropriate secondary antibody. For the primary antibody mouse anti-Pan-neuronal, use donkey anti-mouse 488 as the appropriate secondary antibody. Alternately, use anti-rabbit 488 and anti-mouse 594.</li>
		</ol>
		</li>
		<li>Wash slides three times in PBS for 5 min each.</li>
		<li>Optional: perform nuclear staining.
		<ol>
			<li>Place in Hoechst (or other nuclear stain) at a concentration of 0.03 &#181;g/ml in double distilled H20 for exactly 60 sec.</li>
			<li>Wash slides three times in PBS for 5 min each.</li>
		</ol>
		</li>
		<li>Wash in ddH20.</li>
	</ol>
	</li>
	<li>Coverslipping
	<ol>
		<li>Coverslip slides with an aqueous mounting medium, such as Fluoromount-G or ProlongGold. Take care to remove all bubbles using a cotton-tipped applicator. 
		Note: Other mounting agents could be used, however high bleed-through between dyes has been noted by some within days of coverslipping.</li>
		<li>Use clear nail polish to seal the edges, preventing the sections from drying out due to evaporation. Allow nail polish to dry in a light-tight container while slides remain flat and at room temperature, and then store in a light-tight container wrapped in foil at 4&#160;&#176;C.</li>
	</ol>
	</li>
</ol>
3. Imaging the Stained Tissue
<ol>
	<li>Microscopy
	<ol>
		<li>Allow the nail polish to dry for at least one hour before commencing microscopy, which should take place in a darkened room.</li>
		<li>Acquire photomicrographs using a confocal or research microscope with a fluorescent light source and a digital camera attachment. Using the accompanying software, set the exposure for each wavelength &#8211; 405, 488, and 594 &#8211; separately. Note: in-depth imaging instructions should be available online from the microscope manufacturer.</li>
		<li>Acquire photomicrographs in each channel without moving the sections or adjusting the focus. Take images in color, or alternately in grayscale and convert to color afterward. 
		Note: Color or grayscale images from each channel can be collated in post-processing.</li>
		<li>Ensure that tissue sections are not exposed to ambient light or microscopic light for long periods of time, as photo-bleaching of the sections will occur. To avoid this, increase exposure time rather than increasing light/laser intensity.</li>
		<li>Do not turn off the fluorescent light source within 30 min of being turned on. 
		Note: Switching the source on and off in quick succession may decrease the life span of the fluorescent bulb.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Marrack, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+of+antigens+and+antibodies.">Chemistry of antigens and antibodies.</a> Nature. 134, 468-469 (1934).</li><li>Coons, A. H., Creech, H. J., Jones, R. N., Berliner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+demonstration+of+pneumococcal+antigen+in+tissues+by+the+use+of+fluorescent+antibody.">The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody.</a> J Immunol. 45, 159-170 (1942).</li><li>Marshall, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+adrenocorticotropic+hormone+by+histochemical+and+immunochemical+methods.">Localization of adrenocorticotropic hormone by histochemical and immunochemical methods.</a> The Journal of experimental medicine. 94, 21-30 (1951).</li><li>Mellors, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+demonstration+of+the+in+vivo+localization+of+antibodies:+antigenic+components+of+the+kidney+and+the+pathogenesis+of+glomerulonephritis.">Histochemical demonstration of the in vivo localization of antibodies: antigenic components of the kidney and the pathogenesis of glomerulonephritis.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 3, 284-289 (1955).</li><li>Nakane, P. K., Pierce, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-labeled+antibodies:+preparation+and+application+for+the+localization+of+antigens.">Enzyme-labeled antibodies: preparation and application for the localization of antigens.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 14, 929-931 (1966).</li><li>Avrameas, S., Uriel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+antigen+and+antibody+labelling+with+enzymes+and+its+immunodiffusion+application.">Method of antigen and antibody labelling with enzymes and its immunodiffusion application.</a> Comptes rendus hebdomadaires des seances de l'Academie des sciences. Serie D: Sciences naturelles. 262, 2543-2545 (1966).</li><li>Cuello, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunohistochemistry. , (1983).</li><li>Junqueira, L. C. U., Mescher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Junqueira's basic histology : text and atlas. , (2013).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of visualized experiments : JoVE. , (2012).</li><li>Christensen, N. K., Winther, L., Kumar, G. L., Rudbeck, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Education Guide: Immunohistochemical (IHC) staining methods. , 103-108 (2009).</li><li>Wang, G., Achim, C. L., Hamilton, R. L., Wiley, C. A., Soontornniyomkij, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+method+in+multiple-label+immunofluorescence+confocal+microscopy).">Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy).</a> Methods. 18, 459-464 (1999).</li><li>Feldengut, S., Del Tredici, K., Braak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+sections+of+70-100+mum:+a+novel+technique+and+its+benefits+for+studying+the+nervous+system.">Paraffin sections of 70-100 mum: a novel technique and its benefits for studying the nervous system.</a> Journal of neuroscience methods. 215, 241-244 (2013).</li></ol>

The authors have nothing to disclose.

The overall goal of this communication was to introduce immunohistochemistry procedures to the reader. For this, the example of double-labeling with Iba1 and Pan-neuronal antigens to observe microglia and neurons in paraformaldehyde perfused, sucrose cryoprotected, cryosectioned rat brain was used.
This technique can be adapted to serve endless purposes. An array of different antigens in a variety of tissue types such as, but not limited to brain, lung, liver, kidney and intestine can be visua...

Immunohistochemistry is a process for detecting antigens (i.e. proteins) in tissue sections by using primary antibodies which bind specifically to the antigens of interest. Immunohistochemistry was pioneered by J.R. Marrack in 1934 when he determined that antibodies could localize antigens with great specificity1. Beginning in 1942, some of the first in vitro studies using fluorescent antibodies to visualize immunohistochemistry were published2,3, after which the first in vivo histochemical study was published 4. During the 1960&#8217;s, three decades after the inception of immunohistochemical methods, enzyme-conjugated antibodies began to be used as secondary reagents. These methods were simultaneously and independently developed in France and the U.S.5,6. Today, a wide array of antibodies provides endless possibilities for immunohistochemistry studies7.
The overall goal of this correspondence is to provide a brief introduction into immunohistochemical staining; it is not meant to be a comprehensive and exhaustive review of this technique. In the method outlined, immunohistochemical techniques for two antigens are presented (markers for microglia and neurons) for staining of paraformaldehyde perfused, sucrose cryoprotected, cryosectioned rat brain. Immunohistochemical staining begins with blocking nonspecific antigen binding to reduce background staining. Next, incubation with primary antibody allows for binding to a specific antigen in the tissue. Following the primary antibody, another antibody, termed secondary antibody, is applied to link the primary antibody to a conjugated visualization signal8. The secondary antibody targets the immunoglobulin G (IgG) domain specific to the species in which the primary antibody was raised. The secondary antibody amplifies the signal of the primary antibody since the Fab regions of the secondary antibody bind to multiple sites on the IgG domain of the primary antibody. Either enzymes or fluorescent molecules conjugated to the Fc regions of the secondary antibody enable visualization. For example, a rabbit anti-Iba1 primary antibody is a rabbit IgG molecule specific for Iba1. When donkey anti-rabbit IgG is applied as a secondary antibody, it will recognize and bind to multiple regions of the rabbit anti-Iba1 IgG (see Figure 1). The donkey antibody can be visualized by various methods. This correspondence focuses on detection of a fluorophore conjugated to the secondary antibody, which recognizes the primary antibody, for visualization by fluorescent microscopy. In fluorescent immunohistochemistry, a nuclear stain such as Hoechst or DAPI can be used to visualize all nuclei.
<img alt="Figure 1" src="/files/ftp_upload/52293/52293fig1highres.jpg" /> 
Figure 1: Schematic representation of direct vs. indirect antibody labeling techniques. Antibodies bind to the antigen of interest and can be amplified by secondary antibodies raised against the species of the primary antibodies. This technique can be performed using avidin-biotin complex (ABC) for amplification and DAB for visualization (A), or a directly conjugated fluorescent secondary antibody (B). Alternatively, primary antibodies can be directly conjugated with many different tags, including biotin or a fluorophore (C). <a href="https://www.jove.com/files/ftp_upload/52293/52293fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
An alternative method for visualization of immunohistochemical staining uses 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB; see Figures 1 and 2). This differs from fluorescence by using a biotinylated or horse-radish peroxidase (HRP) conjugated secondary antibody, which provides an enzyme to convert DAB to a precipitate that is visible under bright field microscopy. In instances where a single antigen is of interest or staining is required to be long lasting, DAB may be more appropriate than fluorescent staining. However, DAB staining is not well-suited for differentiation between multiple markers, especially if two nuclear antigens are of interest. For information on DAB materials and protocol modifications, consult Table 1. Alternately, nitro blue tetrazolium chloride/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) can be used to visualize an alkaline phosphatase (AP) conjugated secondary antibody.
<img alt="Figure 2" src="/files/ftp_upload/52293/52293fig2highres.jpg" /> 
Figure 2: Representative images of nickel-enhanced DAB single-labeled rat brain tissue sections. Rat brain sections which are labeled with nickel-enhanced DAB for Iba1 (A) and Pan-neuronal (B) allow for long-lasting analysis of microglia or neurons alone. Scale bar 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52293/52293fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
One must consider the estimated abundance of the antigen of interest within the tissue being analyzed. Indirect methods (as described above) are useful for targets with low abundance. When the antigen of interest is in high abundance, direct methods can be applied. Direct methods involve a primary antibody that is directly conjugated to a visualization signal, and thus no secondary antibody is required. This method simplifies the staining process, but eliminates the amplification achieved by indirect methods. Using a directly conjugated primary antibody also eliminates cross-reactivity of secondary antibodies when double-labeling.
This communication details the protocol for double-labeling with Iba1 and Pan-neuronal (details in Table 1). Iba1 stains microglia in many activation states, including ramified, hyper-ramified, activated, amoeboid, and rod. Pan-neuronal stains neuronal axons, dendrites, and soma. Since Iba1 stains most microglia and Pan-neuronal targets the neuron, this combination of stains is useful in gaining a broad understanding of microglia-neuron interactions.
In sum, immunohistochemical staining relies on the careful selection of antibodies. As the research question becomes more specific, antibodies raised to alternate antigens may be desired. To target a specific microglial activation state, one may elect to use CD45 or CD68 antibodies, rather than Iba1. Further, in working with mice, F4/80 may provide the necessary results. Similarly, neuronal elements can be specifically targeted with antibodies raised against the nucleus, synapse (pre- or post-), axon, and growth cone. Additionally, there are other markers which differentiate the age of the neuron (Double-cortin, NeuN), and neuronal regeneration (GAP-43).

<table border="0" cellpadding="0" cellspacing="0" width="1171">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Name of Material/ Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:523px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand Superfrost Plus Glass Slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>22-034-979</td>
			<td>Used for tissue mounting (1.2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Oven</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:119px;">51028112</td>
			<td>Used for tissue drying (2.1.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mini Pap pen</td>
			<td style="width:132px;">Life Technologies</td>
			<td style="width:119px;">00-8877</td>
			<td>Used in step 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Andwin Scientific Tissue-tek Slide Staining Dish</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">22-149-429</td>
			<td>Used for all washes during staining (2.2), as well as the Hoechst step (2.2.8)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Kimwipes</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">06-666-A</td>
			<td>Used for drying slides (2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Black Staining Box</td>
			<td style="width:132px;">Ted Pella</td>
			<td style="width:119px;">21050</td>
			<td>Used for blocking and staining steps (2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Normal Donkey Serum</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">50-413-253</td>
			<td>Used for block and antibody incubation (2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Mouse &#945;-Pan-neuronal</td>
			<td style="width:132px;">Millipore</td>
			<td style="width:119px;">MAB2300</td>
			<td>Used for primary antibody (2.2.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Rabbit &#945;-Iba1</td>
			<td style="width:132px;">Wako Chemical</td>
			<td style="width:119px;">019-19741</td>
			<td>Used for primary antibody (2.2.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Donkey &#945;-Rabbit 594</td>
			<td style="width:132px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">711-585-152</td>
			<td>Used for secondary antibody (2.2.6)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Donkey &#945;-mouse 488</td>
			<td style="width:132px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">715-545-150</td>
			<td>Used for secondary antibody (2.2.6)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Caterer&#39;s foil</td>
			<td style="width:132px;">Any</td>
			<td style="width:119px;">N/A</td>
			<td>Used in steps 1.2.2 and 2.3.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Fluoromount-G</td>
			<td style="width:132px;">Southern Biotech</td>
			<td style="width:119px;">0100-01</td>
			<td>Used for coverslipping (2.2.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Coverslips</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">12544E</td>
			<td>Used for coverslipping (2.2.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Clear Nail Polish&#160;</td>
			<td style="width:132px;">Any</td>
			<td style="width:119px;">N/A</td>
			<td>Used for coverslipping (2.2.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Axio Observer.Z1 and LSM 710 (laser scanning, confocal)</td>
			<td style="width:132px;">Carl Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td>Used for imaging (3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Axioskop A2</td>
			<td style="width:132px;">Carl Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td>Used for imaging (3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CitriSolv&#160;</td>
			<td>FisherScientific</td>
			<td></td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ABC</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAB Peroxidase kit</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated horse &#945;-rabbit IgG</td>
			<td>Vector Laboratories</td>
			<td>BA-1100</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated horse &#945;-mouse IgG</td>
			<td>Vector Laboratories</td>
			<td>BA-2001</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% Hydrogen Peroxide</td>
			<td>FisherScientific</td>
			<td>H325-500</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Wheaton slide racks and staining dishes</td>
			<td style="width:132px;">TedPella</td>
			<td style="width:119px;">21043</td>
			<td>For DAB protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Masterflex perfusion pump and tubing</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>Used for perfusion (1.1.1 and 1.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Andwin scientific tissue-tek CRYO-OCT compound (case of 12)</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td>Used for tissue freezing (1.2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;">Thermometer (-50 to 50 C)&#160;</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">15-059-228</td>
			<td>Used for tissue freezing (1.2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica&#160;</td>
			<td>CM3500S</td>
			<td>Used for tissue sectioning (1.2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:399px;"></td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Staining Dish, Plastic with 2 Lids</td>
			<td>Grale Scientific</td>
			<td>353</td>
			<td>For antigen retrival</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 Place Staining Rack, Slides Horizontal</td>
			<td>Grale Scientific</td>
			<td>354</td>
			<td>For antigen retrival</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microwave</td>
			<td>Any</td>
			<td>N/A</td>
			<td>For antigen retrival</td>
		</tr>
	</tbody>
</table>

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 staining protocol results in rat brain tissue sections that have microglia fluorescently labeled in the 594 channel (red) and neurons labeled in the 488 channel (green; see Figure 4). If a nuclear stain has been done, it will show in the 405 channel (blue). Images can be taken in different channels and overlaid for direct comparison of the three channels, or between any two channels. Many digital acquisition software suites include this functionality. Double-labeling with Iba1 and Pan-neuronal marke...

Watch this Scientific Journal Video about Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons at JoVE.com

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

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 ...

primer-for-immunohistochemistry-on-cryosectioned-rat-brain-tissue

Research

JoVE Journal

Neuroscience

31.6K Views.  University of Arizona College of Medicine - Phoenix. 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. 

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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.