Name: simple elimination of background fluorescence in formalin-fixed human brain tissue for immunofluorescence microscopy
Uploaded: 2017-09-03T13:00:00
Description: Watch this Scientific Journal Video about Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy at JoVE.com

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 p...

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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.

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			<td>S7653</td>
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			<td>Sarstedt</td>
			<td>82.9923.422</td>
			<td>All components of photobleacher can be substituted based on availability</td>
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			<td>All components of photobleacher can be substituted based on availability</td>
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			<td>Sigma-Aldrich</td>
			<td>C-2404</td>
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			<td>P-7949</td>
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			<td height="40" style="height:40px;">Sodium Hydroxide</td>
			<td>BioShop Canada</td>
			<td>SHY700.1</td>
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			<td>12-587-17B</td>
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			<td height="40" style="height:40px;">Staining Jar</td>
			<td>FisherScientific</td>
			<td>E94</td>
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			<td height="40" style="height:40px;">Orbital Shaker</td>
			<td>Bellco Glass</td>
			<td>&#160;7744-08115</td>
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			<td>Sigma-Aldrich</td>
			<td>T7878</td>
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			<td height="40" style="height:40px;">Bovine Serum Albumin</td>
			<td>FisherScientific</td>
			<td>BP1600-1</td>
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			<td>Aurion</td>
			<td>905.002</td>
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			<td height="40" style="height:40px;">Hydrophobic pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z672548-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)</td>
			<td>ThermoFisher</td>
			<td>MN1020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Mouse Secondary Antibody, Texas Red-X</td>
			<td>ThermoFisher</td>
			<td>T862</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Mouse Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mounting medium</td>
			<td>ThermoScientific</td>
			<td>28-600-42</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass soverslip</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software ZEN 2012 Black Edition 11.0</td>
			<td>Zeiss</td>
			<td>LSM710</td>
			<td>Software accompanies the Confocal Microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RGB Profile Tools macro</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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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