This study was supported in whole or in part by the Canadian Consortium of Neurodegeneration and Aging (CCNA), the Canadian Institute of Health Research (CIHR), the ALS Society of Canada (ALS Canada), and the Alzheimer Society of Canada (ASC). The authors would like to thank Sultan Darvesh and Andrew Reid from the Maritime Brain Tissue Bank for providing the FTLD brain tissues, Milan Ganguly from the Spatio-Temporal Targeting and Amplification of Radiation Response (STTARR) program and its affiliated funding agencies for tissue embedding and sectioning services, and the Advanced Optical Microscopy Facility (AOMF) for providing microscopy instruments. Kevin Hadley is thanked for critical review of the manuscript.

Note: The work presented was performed in compliance with recognized international standards, including the International Conference on Harmonization (ICH), the Council for International Organizations of Medical Sciences (CIOMS), and the principles of the Declaration of Helsinki. Use of human tissue was with the approval of University Health Network Research Ethics Board. The human brain samples were collected as a part of the Maritime Brain Tissue Bank. At the time of collection, informed consent was obtained from all patients.
1. Construction of photobleaching apparatus and solutions
<ol>
	<li>Prepare stock solutions.
	<ol>
		<li>Prepare 1 L of 1x stock Tris-buffered saline (1x TBS) solution (150 mM NaCl, 50 mM Tris-Cl, pH 7.4) by dissolving 8.77 g of NaCl and 6.06 g of Tris base in 800 mL of ddH2O and adjust the pH to 7.4 using HCl. Bring up the volume to 1 L and autoclave.</li>
		<li>Prepare 10% (200x stock) sodium azide by dissolving 1 g of sodium azide in 10 mL of ddH2O (10%, 200x stock).</li>
	</ol>
	</li>
	<li>For 1-3 standard size slides, use a single 100 mm x 100 mm transparent, square petri dish as a slide chamber. For a single slide chamber, add 0.25 mL of 200x sodium azide stock to 50 mL of 1x TBS to make a 0.05% azide-TBS solution. Stack 2-3 slide chambers vertically to process additional slides. Prepare an additional 50 mL of azide-TBS solution for each chamber.</li>
	<li>Create a scaffold to elevate the slide chamber(s) such that a lamp head can fit underneath.
	<ol>
		<li>For a 100 mm x 100 mm slide chamber, cut openings in the bottom and sides of a 100 mm x 100 mm x 30 mm plastic food container and invert the container. Ensure the side openings are large enough to fit an LED light source and ensure the bottom opening is large enough such that light from the light source reaches the sample chamber without impediment.</li>
		<li>Apply electrical tape to the scaffold to increase the grip between the scaffold and the sample chamber/benchtop. Use any alternative materials to construct the scaffold so long as it securely elevates the slide chamber without impeding the light from reaching the sample.</li>
	</ol>
	</li>
	<li>Remove any diffusers or opaque plastic from the desk lamp that may impede the LED light from directly reaching the sample (if possible) and orient the LED array upwards. Place the scaffold and slide chamber(s) above the LED array. Use a lamp with a flexible neck for easy manipulation.</li>
	<li>Construct a reflective dome cover for the apparatus by lining the inside of a box large enough to cover the slide chamber and scaffold with aluminum foil. Use a 1 mL pipette tip box for a single chamber or a 150 mm x 150 mm x 150 mm cardboard box for multiple, vertically stacked chambers.</li>
</ol>
2. Photobleaching pre-treatment of tissue sections
NOTE: Tissue section preparation may vary depending on the source of tissue and fixation and embedding methods used. Here, brain tissue (orbitofrontal gyri) from a case of FTLD-T was fixed for ~2 days in formalin, run through a sucrose gradient, embedded in OCT, and cut to 10 &#181;m thick sections using a cryostat.
<ol>
	<li>In a 4 &#186;C cold room, cold cabinet, or refrigerator, orient the lamp under the scaffold and place the sample chamber on the scaffold. Pour 50 mL of azide-TBS solution into the sample chamber.</li>
	<li>Submerge tissue sections mounted on standard glass microscope slides into the slide chamber containing azide-TBS using clean forceps. For multiple slides, ensure that the slides are placed in the chamber on a single layer.</li>
	<li>Cover the apparatus with the reflective dome, turn on the LED lamp, and incubate for 48 h at 4 &#186;C.</li>
</ol>
3. Immunofluorescence
<ol>
	<li>To stain the tissue for phosphorylated tau using DAPI counterstain and Alexa 488- and Texas Red-conjugated secondary antibodies, first prepare solutions for antigen retrieval, permeabilization, blocking, and primary antibody binding.
	<ol>
		<li>Prepare 500 mL of antigen retrieval buffer (10 mM citric acid, 2 mM ethylenediaminetetraacetic acid, 0.05% Tween 20; pH 6.2) by dissolving 0.92 g of citric acid and 0.37 g of ethylenediaminetetraacetic acid (EDTA) in 500 mL of ddH2O. Adjust the pH to 6.2 with NaOH and add 0.25 mL of Tween 20.</li>
		<li>Prepare 500 mL of 0.025% Triton X-100 in TBS solution (TBS-Triton) by adding 0.125 mL of Triton X-100 to 500 mL 1x TBS.</li>
		<li>Prepare a 1% bovine serum albumin (BSA) solution in TBS (BSA-TBS buffer) by dissolving 0.1 g BSA in 10 mL TBS.</li>
		<li>Prepare a blocking solution by adding 0.2 mL of normal goat serum to 1.8 mL of 1% BSA/TBS.</li>
		<li>For each slide, prepare 150 &#181;L of primary antibody solution (1:100 dilution) by pipetting 1.5 &#181;L of anti-phospho-PHF-tau pSer202 + Thr205 (AT8) antibody into 148.5 &#181;L of 1% BSA-TBS buffer and leave on ice.</li>
	</ol>
	</li>
	<li>To perform antigen retrieval, submerge the photobleached slides vertically in a slide collector containing 25 mL of antigen retrieval buffer. Secure the collector with tape and/or strings such that the collector does not fall into the water bath. Heat the collector in a water bath at 90 &#186;C for 30 min and allow the collector to cool to room temperature for 30 min before removing the slides. Do not remove the slides immediately as it will cause the sections to dry out.</li>
	<li>Transfer the slides from the antigen retrieval collector into a staining jar filled with 30 mL of TBS-Triton and wash the sections for 5 min on an orbital shaker with gentle shaking. Repeat the wash once with fresh TBS-Triton. Wick away excess buffer with a lint-free tissue and outline the tissue with a hydrophobic pen. Take care not to let the slides dry out.
	<ol>
		<li>For each slide, block the tissue by pipetting 200 &#181;L of blocking solution onto the tissue and place the slide in a humidified chamber. Construct the chamber by placing a slide rack inside a pipette tip box containing a wet paper towel. Incubate at room temperature for 2 h on a level surface. Ensure that the blocking solution fully covers the tissue.</li>
	</ol>
	</li>
	<li>Remove the blocking solution by aspiration and pipette 100-150 &#181;L of primary antibody solution onto the tissue. Ensure sufficient volume of antibody is present and that the section is on a level surface to avoid pooling of antibody solution to one side. Incubate at 4 &#186;C overnight in a humidified chamber.</li>
	<li>Prepare the secondary antibody mixture and the DAPI nuclear counterstain.
	<ol>
		<li>For each slide, prepare 150 &#181;L of secondary antibody mixture (1:100 dilutions) by adding 1.5 &#181;L of goat anti-mouse Alexa 488 and 1.5 &#181;L of goat anti-mouse Texas Red to 147 &#181;L of BSA-TBS and leave on ice.</li>
		<li>Prepare 0.1 &#181;g/mL DAPI counterstain by serial dilution. Mix the stock solution thoroughly and dilute 1 &#181;L of stock 5 mg/mL DAPI in 999 of TBS to make 1 mL of 5 &#181;g/mL solution. For each slide, dilute 3 &#181;L of 5 &#181;g/mL solution with 147 &#181;L of TBS to a final concentration of 0.1 &#181;g/mL. 
		CAUTION: DAPI is a known mutagen and should be handled with care.</li>
	</ol>
	</li>
	<li>Remove the primary antibody by aspiration. Submerge the slides in a glass staining jar containing 30 mL of TBS-Triton and wash for 5 min with gentle mixing on an orbital shaker. Repeat the wash step with fresh TBS-Triton. Wick away excess TBS-Triton and pipette 100 to 150 &#181;L of secondary antibody mixture to each slide.
	<ol>
		<li>Ensure the tissue is fully covered by the antibody mixture. Incubate for 2 h at room temperature in the humidified chamber in the dark.</li>
	</ol>
	</li>
	<li>Remove the secondary antibody mixture by aspiration and transfer the slide into a glass staining jar containing 30 mL of TBS (no Triton). Wash for 5 min with gentle mixing on an orbital shaker. Repeat the wash step with fresh TBS. Apply 100 to 150 &#181;L of 0.1 &#181;g/mL DAPI counterstain to each slide and incubate for 10 min at room temperature in the dark.</li>
	<li>Transfer the slides to a glass staining jar containing TBS and wash 3 times with gentle mixing for 5 min each, using fresh TBS for each wash. Wick away excess buffer.</li>
	<li>Apply 3 drops of aqueous mounting medium to the tissue. Using forceps, gently lower a clean glass coverslip onto the tissue, starting with one edge and slowly lowering the other edge to avoid trapping of air bubbles. Take care not to dislodge the coverslip if imaging immediately. Otherwise, allow the mounting medium to dry before storing at 4 &#186;C in the dark.</li>
</ol>
4. Fluorescence microscopy
<ol>
	<li>Turn on the fluorescence lamp, the microscope and the computer and allow the lamp to warm up for 15 min. Place the stained tissue slides in the fluorescence microscope stage. Use the bright field image to locate the tissue at 10x magnification.</li>
	<li>Apply a drop of ddH2O to the coverslip surface and use a 20x water immersion objective lens (NA=1.0). Select the 4-line average plane scan setting. Set the pinhole size to 1 Airy unit that gives an optical slice of ~3 microns. Select the laser excitation and emission wavelengths for each fluorophore in separate tracks for best signal. 
	NOTE: Alexa 488: &#955;ex = 488 nm (argon laser) &#955;em = 493-570 nm; Texas Red: &#955;ex = 561 nm (DPSS 561 nm laser), &#955;em = 601-635 nm; DAPI: &#955;ex = 405 nm (Diode 405 laser), &#955;em = 410-507 nm.
	<ol>
		<li>Adjust the laser power and gain settings to optimize the signal intensity for each track. Collect the composite image and save. Use the same laser settings to compare fluorescence intensities in a different slide.</li>
	</ol>
	</li>
	<li>For visualization of fluorescence intensity in each channel, install the RGB profile tools macro for ImageJ10. Save the macro from the webpage as a text file (https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt). From the ImageJ menu, select Plugins -&#62; Macros -&#62; Install; select the text file to install the RGB profiles tool.
	<ol>
		<li>Open the confocal image file in ImageJ and convert the composite images from the 3 stacks to RGB by performing the following: Image -&#62; Color -&#62; Channel Tool. Select &#34;Composite&#34; from the dropdown menu and check all three channels. Then, select Image -&#62; Type -&#62; RGB color.</li>
		<li>&#160;Select the RGB profile tools icon and draw a line across the section in the image to be profiled. Save the intensity data as a spreadsheet for plotting.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Banerjee, B., Miedema, B. E., Chandrasekhar, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+basement+membrane+collagen+and+elastin+in+the+autofluorescence+spectra+of+the+colon.">Role of basement membrane collagen and elastin in the autofluorescence spectra of the colon.</a> J Investig Med. 47 (6), 326-332 (1999).</li><li>Terman, A., Brunk, U. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofuscin+.">Lipofuscin .</a> Int J Biochem Cell Biol. 36 (8), 1400-1404 (2004).</li><li>Del Castillo, P., Llorente, A. R., Stockert, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+fixation,+exciting+light+and+section+thickness+on+the+primary+fluorescence+of+samples+for+microfluorometric+analysis.">Influence of fixation, exciting light and section thickness on the primary fluorescence of samples for microfluorometric analysis.</a> Basic Appl Histochem. 33 (3), 251-257 (1989).</li><li>Ottis, P., Koppe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+rat+brain+lipofuscin+proteome.">Human and rat brain lipofuscin proteome.</a> Proteomics. 12 (15-16), 2445-2454 (2012).</li><li>Sun, Y., Chakrabartty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+elimination+of+lipofuscin+fluorescence+from+formalin-fixed+brain+tissue+by+white+phosphor+light+emitting+diode+array.">Cost-effective elimination of lipofuscin fluorescence from formalin-fixed brain tissue by white phosphor light emitting diode array.</a> Biochem Cell Biol. 94 (6), 545-550 (2016).</li><li>Davis, A. S., Richter, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+Diminishing+Autofluorescence+in+Formalin-fixed+Paraffin-embedded+Human+Respiratory+Tissue.">Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue.</a> J Histochem Cytochem. 62 (6), 405-423 (2014).</li><li>Zimmermann, T., Rietdorf, J., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+imaging+and+its+applications+in+live+cell+microscopy.">Spectral imaging and its applications in live cell microscopy.</a> FEBS Lett. 546 (1), 87-92 (2003).</li><li>Duong, H., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multispectral+LED+array+for+the+reduction+of+background+autofluorescence+in+brain+tissue.">A multispectral LED array for the reduction of background autofluorescence in brain tissue.</a> J Neurosci Methods. 220 (1), 46-54 (2013).</li><li>Albeanu, D. F., Soucy, E., Sato, T. F., Meister, M., Murthy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+arrays+as+cost+effective+and+efficient+light+sources+for+widefield+microscopy.">LED arrays as cost effective and efficient light sources for widefield microscopy.</a> PLoS One. 3 (5), 1-7 (2008).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Robertson, J. B., Zhang, Y., Johnson, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+diode+flashlights+as+effective+and+inexpensive+light+sources+for+fluorescence+microscopy.">Light-emitting diode flashlights as effective and inexpensive light sources for fluorescence microscopy.</a> J Microsc. 236 (1), 1-4 (2009).</li><li>Mackenzie, I. R. A., Neumann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+neuropathology+of+frontotemporal+dementia:+insights+into+disease+mechanisms+from+postmortem+studies.">Molecular neuropathology of frontotemporal dementia: insights into disease mechanisms from postmortem studies.</a> J Neurochem. 138 (S1), 54-70 (2016).</li><li>Kouri, N., Whitwell, J. L., Josephs, K. A., Rademakers, R., Dickson, D. 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The authors have nothing to disclose.

The photobleaching pre-treatment of tissues described in this manuscript allows for effective elimination of autofluorescence using off-the-shelf components. The protocol describes immunofluorescence imaging of phosphorylated tau aggregates in formalin-fixed human brain tissue using secondary antibodies conjugated to Alexa 488 and Texas Red, with DAPI as a nuclear counterstain. To apply the method to other tissues, we recommend performing a 48 h photobleaching pre-treatment to the sample as a starting point. After photob...

Fluorescence microscopy using antibodies targeting specific proteins is routinely used to visualize proteins of interest in cell culture and tissues. A major complication to the acquisition of clear and definitive images in immunofluorescence is autofluorescence, which can be caused endogenously in mammalian tissue by the age pigment lipofuscin and by proteins such as elastin and collagen1,2. Other sources of autofluorescence can be introduced through sample preparation steps such as aldehyde fixation3. Lipofuscin granules, composed primarily of oxidatively modified protein and lipid degradation residues, accumulate in long-living cells with increased age2. This causes difficulties in imaging postmitotic tissues such as the brain and cardiac or skeletal muscles, as the fluorescence emission spectrum of lipofuscin is broad and variable, often coinciding with the emission wavelength of common fluorophores used for labeling4. These factors make imaging of human brain tissue from cases of late-onset neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) especially challenging.
To reduce autofluorescence, we have devised a technique in which we irradiate the slide-mounted tissue sections with a white light emitting diode (LED) array using a household desk lamp5. This simple technique provides an alternative to techniques that use chemical quenchers such as CuSO4 in ammonium acetate, or commercially available quenching dyes such as Sudan Black B and Eriochrome Black T6. It also has significant cost-saving over multispectral LED lamp photobleaching techniques and avoids complications and artefacts generated from digital autofluorescence removal methods such as spectral un-mixing7,8. White phosphor LEDs have a broad emission spectrum, high luminosity and low manufacturing cost, making them ideal as an off-the-shelf component for photobleaching a variety of chromophores5,9.
In this protocol, we demonstrate the construction of a photobleaching apparatus using accessible components and apply photobleaching to a case of FTLD tissue containing tau-positive inclusions (FTLD-T) using an antibody specific for phosphorylated tau. We demonstrate the effect of photobleaching on imaging fluorescently-labeled antibodies employing two commonly-used chromophores: Alexa 488 and Texas Red. The effect of photobleaching versus untreated sections or those treated with a commercial chemical quencher are quantified and compared. This photobleaching pre-treatment can be incorporated into any standard immunofluorescence staining protocol to remove autofluorescence in a biological sample.

<table><tbody><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>T6066</td><td></td></tr><tr><td>Sodium Choloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Caledon Laboratory Chemicals</td><td>1506656</td><td></td></tr><tr><td>Sodium Azide</td><td>BioShop Canada</td><td>SAZ001</td><td></td></tr><tr><td>100 mm x 100 mm x 20 mm Pitri dish</td><td>Sarstedt</td><td>82.9923.422</td><td>All components of photobleacher can be substituted based on availability</td></tr><tr><td>6 W LED Dimmable Desk Lamp</td><td>DBPower</td><td>DS501</td><td>All components of photobleacher can be substituted based on availability</td></tr><tr><td>Citric Acid</td><td>Sigma-Aldrich</td><td>C-2404</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;(EDTA)</td><td>BioShop Canada</td><td>EDT001</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P-7949</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>BioShop Canada</td><td>SHY700.1</td><td></td></tr><tr><td>Water bath</td><td>Haake Fisons</td><td>K15</td><td></td></tr><tr><td>Slide collector</td><td>FisherScientific</td><td>12-587-17B</td><td></td></tr><tr><td>Staining Jar</td><td>FisherScientific</td><td>E94</td><td></td></tr><tr><td>Orbital Shaker</td><td>Bellco Glass</td><td>&amp;nbsp;7744-08115</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T7878</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>FisherScientific</td><td>BP1600-1</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Aurion</td><td>905.002</td><td></td></tr><tr><td>Hydrophobic pen</td><td>Sigma-Aldrich</td><td>Z672548-1EA</td><td></td></tr><tr><td>Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)</td><td>ThermoFisher</td><td>MN1020</td><td></td></tr><tr><td>Goat anti-Mouse Secondary Antibody, Texas Red-X</td><td>ThermoFisher</td><td>T862</td><td></td></tr><tr><td>Goat anti-Mouse Secondary Antibody, Alexa Fluor 488</td><td>ThermoFisher</td><td>A-11029</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Mounting medium</td><td>ThermoScientific</td><td>28-600-42</td><td></td></tr><tr><td>Glass soverslip</td><td></td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM710</td><td></td></tr><tr><td>Imaging software ZEN 2012 Black Edition 11.0</td><td>Zeiss</td><td>LSM710</td><td>Software accompanies the Confocal Microscope</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/download.html</td></tr><tr><td>RGB Profile Tools macro</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt</td></tr><tr><td>Commercial chemical quencher</td><td>Biotum</td><td>23007</td><td></td></tr></tbody></table>

simple elimination of background fluorescence in formalin-fixed human brain tissue for immunofluorescence microscopy

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue sample. A great hindrance to the acquisition of high quality immunofluorescence images is endogenous autofluorescence of the tissue caused by aging pigments such as lipofuscin or by common sample preparation processes such as aldehyde fixation. This protocol describes how background fluorescence can be greatly reduced through photobleaching using white phosphor light emitting diode (LED) arrays prior to treatment with fluorescent probes. The broad-spectrum emission of white phosphor LEDs allow for bleaching of fluorophores across a range of emission peaks. The photobleaching apparatus can be constructed from off-the-shelf components at very low cost and offers an accessible alternative to commercially available chemical quenchers. A photobleaching pre-treatment of the tissue followed by conventional immunofluorescence staining generates images free of background autofluorescence. Compared to established chemical quenchers which reduced probe as well as background signals, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. Although photobleaching requires more time for pre-treatment, higher intensity LED arrays may be used to reduce photobleaching time. This simple method can potentially be applied to a variety of tissues, particularly postmitotic tissues that accumulate lipofuscin such as the brain and cardiac or skeletal muscles.

The photobleaching pre-treatment step can be added to a standard immunofluorescence protocol immediately prior to antigen retrieval and immunostaining (Figure 1A). Assembly of the photobleaching apparatus can also be performed using various, inexpensive, off-the-shelf components (Figure 1B). The emission spectrum of white phosphor LEDs covers a wide range of wavelengths which makes them suitable for broad-range photobleaching, ag...

Watch this Scientific Journal Video about Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy at JoVE.com

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining.

Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue ...

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Biochemistry

17.1K Views.  University of Toronto. Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining. 

Video: Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Neuroscience

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 ...

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 ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Immunology and Infection

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

High-Resolution 3D Reconstruction of Human Brain with SHORT and LSFM

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Author Spotlight: Advancing 3D Cytoarchitecture Analysis - Rapid Volumetric Reconstruction of the Human Brain 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its ...

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

Biology

Immunohistological Detection of Pathogens in Brain Tissue

Take a formalin-fixed human brain tissue embedded in paraffin. The tissue exhibits pathogenic microbes present both inside the cells and extracellularly.
Wash to remove the paraffin, then rehydrate the tissue using decreasing alcohol concentrations.
Heat with an acidic buffer to break the formalin-induced protein crosslinks, unmasking antigenic epitopes on the bacteria.
Incubate with ammonium chloride to neutralize residual reactive aldehyde groups formed during fixation, preventing non-specific interaction with antibodies.
Treat with a detergent to permeabilize cellular membranes, facilitating antibody penetration.
Apply a blocking solution to prevent non-specific antibody binding.
Incubate with primary antibodies that target extracellular and intracellular microbes.
Apply fluorophore-conjugated secondary antibodies that bind to the primary antibodies.
Add a fluorescent DNA-binding dye to stain the nuclei.
Treat with an autofluorescence eliminator to quench fluorescence from autofluorescent intracellular granules, minimizing background signals.
Using fluorescence microscopy, examine the tissue to confirm the presence of microbes.

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

Summary

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Chapters in this video

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

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

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

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Free-floating Immunostaining of Mouse Brains

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistological Detection of Pathogens in Brain Tissue