We thank Ms. Bonnie Phetteplace, RN for assistance during video graphing the FNAB procedure demonstration.

Introduction
Systemic amyloidosis is a highly variable disease that refers to extracellular deposition of various proteins that are collectively referred to as amyloid. Deposition of these amyloid fibrils with beta pleated configuration leads to various clinical presentations depending on the organs involved. The most clinically relevant manifestations of systemic amyloidosis are noted when there is involvement of critical organs such as the heart, liver, and/or kidney. Historically, these involved organs would be biopsied to demonstrate the presence of amyloid. These moderately invasive procedures carried a significant risk including hemorrhage. Fat pad aspiration has since been shown to provide a reliable and noninvasive method for the detection of amyloid in systemic amyloidosis 1. This procedure is essentially comparable to the liposuction of the subcutaneous fat in the anterior abdominal wall under local anesthesia to retrieve fibroadipose tissue for evaluating scant amyloid deposits in small blood vessel walls 2.
The most frequently utilized method for amyloid identification in tissue remains the characteristic apple-green birefringence pattern seen when Congo red stained sections are visualized under a polarized light microscope 3. When fat pad aspiration is performed, Congo red stains can be done on either direct smeared slides or cell block preparations of the aspirated adipose tissue. However, patients in early stages of amyloidosis have scant amyloid deposits, which greatly reduces the sensitivity of Congo red stained cell block sections 4,5. Ultrastructural evaluation of fat pad aspirates by electron microscopy has better reproducibility and improved sensitivity 4. Therefore, it is recommended to submit all fat pad aspirates for both preparation of a cell block and for performing electron microscopy 2.
Fat pad aspiration is a relatively low cost and noninvasive method for obtaining tissue to diagnose systemic amyloidosis. This article describes fat pad aspiration procedure along with details about sample processing to submit specimen for both Congo red staining and ultrastructural evaluation by electron microscopy. In this video, we demonstrate this reproducible and simple procedure to retrieve optimal diagnostic material.

1. Performing the FNA of Anterior Fat Pad
A. Anesthetization of the local area (Figure 1 &amp; 2)
<ol>
	<li>Using alcohol swabs or the skin cleansing agent preferred by a particular institution, clean the skin on the lower quadrant area of the abdomen lateral to the midline and below the umbilicus (Figure 1).</li>
	<li>Mark a rhomboid shaped area about 2 x 2 inches as shown in Figure 1 with a marking pen.</li>
	<li>Aspirate approximately 10 mL of 1% lidocaine with an 18G needle. Attach a 25G 1 ½ inch needle to the syringe. Remove any trapped air by tapping the upright syringe while pressing the plunger until liquid is dispensed and no air bubbles are present.</li>
	<li>Anesthetize along the borders of the rhomboid area already marked as shown in Figure 2. Start by inserting the 25G needle just under the skin at point A and carefully push the needle subcutaneously to point X. Pull back on the syringe plunger to ensure that you are not within a vessel. Then slowly push the plunger to infiltrate up to 2.5 mL of lidocaine (about ¼ of the lidocaine in 10 mL syringe) while withdrawing the needle to point A without letting the needle come out of the skin at point A (Figure 2a3). With the needle still under the skin change the direction towards point Y (Figure 2a4) and push the needle subcutaneously to point Y (Figure 2b1). As before, confirm that the needle is not in a vessel and dispense approximately 2.5 mL of lidocaine while slowly withdrawing the needle through point A (Figure 2b3 through 2b4). Repeat similar steps starting from point B and dispensing lidocaine subcutaneous from points B to X and B to Y (Figure 2c1 through 2d4). Prevent bleeding from the pricks A and B by firm application of sterile gauge piece.</li>
	<li>You may now check that the area is anesthetized by lightly touching the skin within the anesthetized rhomboid by tip/corner of cotton gauze piece, by comparing with adjacent unanesthetized skin.</li>
</ol>
B. Performing fat pad aspiration (Figure 3)
(Application of local anesthesia prior to performance of fat pad aspiration may be bypassed, depending on the regional and individual preferences. Properly performed FNAB procedure for anterior fat pad aspiration can be completed in one prick. However, depending on the pain threshold of individual patient, maneuvering of 18G needle back and forth in to the subcutaneous fat tissue is relatively distressing. The anesthetization method described here achieves the effect in only two pricks with 25G needle and averts the pain with improved tolerance to the procedure.)
<ol>
	<li>Assemble 18G 1 ½ inch needle onto a 10 mL syringe. Mount the syringe-needle assembly in the syringe grip ("FNAB grip/gun") for proper application and release of vacuum.</li>
	<li>Insert the tip of the needle into the subcutaneous fat within the cleansed and anesthetized rhomboid area (Figure 3a).</li>
	<li>Fully retract the plunger of the syringe with the needle inserted in the subcutaneous tissue to generate vacuum (Figure 3b).</li>
	<li>Maintain the vacuum and maneuver the needle back and forth in various directions in to the subcutaneous fat (Figure 3c through 3i). Each stroke should be as long as possible with the length of the needle selected without letting the needle come out of the skin. Maximum sampling is achieved by changing direction with each stroke (Figure 3i). It is important to keep the direction of the needle tangential to the serosa and parallel to the skin surface to avoid puncturing of the peritoneal cavity.</li>
	<li>The fibroadipose tissue accumulates in the syringe. Once enough fibroadipose tissue fragments (up to 1 mL of fragments rich blood mixed specimen) are retrieved, release the vacuum completely and remove the needle (Figure 3k).</li>
	<li>Have the patient or the assistant apply firm pressure on the area of procedure with a pad of gauze to prevent extravasation of blood.</li>
</ol>
2. Specimen Processing
<ol>
	<li>Place at least 5-6 fragments of fibroadipose tissue in glutaraldehyde solution for electron microscopy (Figure 4B).</li>
	<li>Depending on the institutional protocol, a few smears of the fibroadipose tissue fragments may be prepared by spreading them between two slides (Figure 4A).</li>
	<li>The remaining material is allowed to clot in the syringe (this may take 5-7 minutes, depending on the clotting time) (Figure 4C).</li>
	<li>Aspirate 10% formalin in the syringe so that the clotted fibroadipose tissue material is dislodged from the wall of the syringe and free floating (Figure 4C2). Remove the plunger from the syringe (Figure 4C3) and transfer the clotted fibroadipose tissue into the 10% formalin container from the open end of the syringe opposite the nozzle end (Figure 4C4).</li>
	<li>Label the containers appropriately and submit the glutaraldehyde for electron microscopy and the formalin for H &amp; E section and Congo red stain for evaluation under polarizing microscopy. If smears are prepared, they may be processed according to the protocol of the individual laboratory.</li>
</ol>
<img src="/files/ftp_upload/1747/1747fig1.jpg" alt="Abdominal region anatomy diagram with labeled quadrants and reference grid." data-altupdatedat="1747911167000" data-alt="Figure 1"> Figure 1. Area to be anesthetized for FNAB of anterior fat pad.
<img src="/files/ftp_upload/1747/1747fig2.jpg" alt="Blood collection diagram; lancet use; step-by-step sequence; safe sampling process; medical guide." data-altupdatedat="1747911167000" data-alt="Figure 2"> Figure 2. Anesthetization of the local area.
<img src="/files/ftp_upload/1747/1747fig3.jpg" alt="Fine-needle aspiration diagram; tissue sampling process, vacuum syringe, needle insertion steps." data-altupdatedat="1747911167000" data-alt="Figure 3"> Figure 3. Performing FNAB of anterior fat pad.
<img src="/files/ftp_upload/1747/1747fig4.jpg" alt="Direct cytology smear and electron microscopy preparation diagram; fibroadipose tissue processing steps." data-altupdatedat="1747911167000" data-alt="Figure 4"> Figure 4. Processing of anterior fat pad aspirate to be submitted to laboratory for detecting amyloid deposits.
3. Representative Results
<img src="/files/ftp_upload/1747/1747fig5.jpg" alt="Histology, tissue section, stained microscopy image, cellular structure analysis." data-altupdatedat="1747911167000" data-alt="Figure 5"> Figure 5. Amyloid in the wall of small blood vessels in fibroadipose tissue fragments. Fibroadipose tissue was formalin-processed, paraffin embedded, stained with Congo red, and examined by polarizing light microscopy. Amyloid in the wall of small blood vessels shows apple green birefringence (white arrow).
<img src="/files/ftp_upload/1747/1747fig6.jpg" alt="Histology slide, polarized light microscopy; fibrous tissue sample with birefringence analysis." data-altupdatedat="1747911167000" data-alt="Figure 6"> Figure 6. Tissue lacking amyloidosis. The apple green birefringence is absent in tissues of a different patient without amyloidosis. Blue birefringence (white arrow) of collagen fibers, are usually present in almost all specimens.
<img src="/files/ftp_upload/1747/1747fig7.jpg" alt="Nanostructure observed via electron microscopy; surface texture at 1200000x magnification." data-altupdatedat="1747911167000" data-alt="Figure 7"> Figure 7. Fibrils consistent with amyloid in the blood vessel wall. Electron micrograph of straight, non-branching, randomly-scattered 8-10 nm diameter fibrils formed by amyloid in the blood vessel wall.

<ol>
	<li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+diagnosis+of+systemic+amyloidosis.">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li><li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates:+Is+Congo+red+sufficient?.">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?.</a> Amyloid. , .</li><li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Amyloidosis.">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li><li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis:+How+can+the+clinical+role+of+the+test+be+improved?.">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?.</a> Mod Pathol. 20, 1A-380A (2007).</li><li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+typing+of+systemic+amyloidosis:+The+role+of+abdominal+fat+pad+fine+needle+aspiration.">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li><li>Hazenberg, I. I., Rijswijk, C. V. a. n., H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice.">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li><li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis:+specificity,+positive+predictive+value,+and+diagnostic+pitfalls.">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li><li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+Amyloidosis.">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li><li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence.">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li><li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+amyloid+in+fat+pad+aspiration:+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy.">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li><li>DeMay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+Needle+Aspiration+Biopsy.">Fine Needle Aspiration Biopsy.</a> The Art &#38; Science of Cytopathology. , 465-46 (1996).</li><li>Arbustini, E., Vrga, L., Concardi, M., Pallandini, G., Obici, L., Merlini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+and+Immuno-electron+Microscopy+of+Abdominal+Fat+Identifies+and+Characterizes+Amyloid+Fibrils+in+Suspected+Cardiac+Amyloidosis.">Electron and Immuno-electron Microscopy of Abdominal Fat Identifies and Characterizes Amyloid Fibrils in Suspected Cardiac Amyloidosis.</a> Amyloid. 9, 108-114 (2002).</li></ol>

No conflicts of interest.

Diagnosis of amyloidosis is usually achieved with a tissue biopsy of the affected organs such as the kidney, liver, and/or heart. This approach has high diagnostic yield, however, is invasive and may be associated with complications including hemorrhage 2. Rectal, gingival, and bone marrow biopsies were preferred for diagnosis as relatively less invasive approach in 1960s 6,7,8. Abdominal fat pad aspiration was reported in 1973 as a safe, minimally invasive, simple, and less expensive procedure for the tissue diagnosis of systemic amyloidosis 1. However, the details of the procedure and approach to processing of the retrieved specimen is not coherently reported. This video and the article describe the procedure step by step.
The approach for detecting amyloid in fat pad aspiration, similarly, is also not well standardized with a few studies comparing and evaluating various approaches 1,5,6,7,8. Evaluation of anterior fat pad aspirates with Congo red staining is less sensitive with lower interobserver reproducibility especially in early cases of amyloidosis with scant amyloid deposits 5. Immunohistochemistry performed on formalin fixed paraffin-embedded cell block sections and Congo red fluorescence performed on cytology smears have been reported to improve the diagnostic sensitivity 9,10. Electron microscopy improves the detection and identification of scant amyloid fibrils in small blood vessel walls in the fibroadipose tissue in fat pad aspirates 4, 5. Based on this information, this article describes processing of the specimen to prepare cell blocks (for evaluation of Congo red stained cell block sections under polarizing microscope for diagnostic apple green birefringence and also for immunohistochemistry as indicated) and for electron microscopy. However, depending on the approach selected for evaluation of the specimen for amyloid, it may be subjected to cytology smear preparation, cell block preparation, and processing for electron microscopy. Some laboratories perform the tests on the smears prepared from the aspirated fibroadipose tissue fragments and stained with Congo red to study the fluorescence 10.
Generally in a late amyloidosis, the light microscopy with Congo red polarizing microscopy may be sufficient; however, in early stages of the disease Congo red staining with polarizing microscopy on cell block sections is less sensitive and reliable for fat pad aspirates 4,5,11. Other special stains including Thioflavin T may be used with similar limitations. Other approaches to detect amyloid include evaluation of the aspirate smears for amyloid with Congo red polarizing microscopy and fluorescence 10. In our experience this method is less reproducible. Evaluation of the blood vessels in fat pad for amyloid by electron microscopy overcomes these limitations 4,11. Further characterization of amyloid has been reported by immunoelectron microscopy 12; however, these methods may not be available widely in a non-tertiary care setting.
Needles used for performing aspiration procedures may be categorized according to their gauge sizes into fine (21-25G), intermediate (18-20G), and large (eg- 14G) 11. Anterior fat pad aspirations have been reported to be performed by using variable gauges ranging from intermediate (18 to 20G) to fine (21 to 22G) 2,10. Most of the mass lesions are aspirated with finer gauge needles (such as 25G) to obtain good cytology smears along with additional passes with wider gauge needle (such as 18G) to retrieve additional sample for cell blocks.
During anterior fat pad aspiration, the cohesive fibroadipose tissue does not aspirate well with finer needles. Cell block preparation and electron microscopy are important for evaluation of small blood vessel walls in fibroadipose tissue fragments in the fat aspirates. Wider gauge needles (such as 18G) are recommended to yield diagnostic adequate material 2,10. As the procedure is comparable to the conventional FNAB, fat pad aspiration is referred to as FNAB even though wider gauge needles may be used for performing it 5.

A correction was made to <a href="https://www-jove-com.remotexs.ntu.edu.sg/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

<table><tbody><tr><td>Alcohol swabs</td><td></td><td></td><td></td></tr><tr><td>gauze pads</td><td></td><td></td><td></td></tr><tr><td>marking pen</td><td></td><td></td><td></td></tr><tr><td>10 mL syringes x2</td><td></td><td></td><td></td></tr><tr><td>1% lidocaine</td><td></td><td></td><td>local anesthetic- If lidocaine is used alone, it causes an initial burning sensation after instillation. This can be prevented by using a 1:1 mixture of 1% lidocaine and 1% sodium-bi-carbonate</td></tr><tr><td>18 gauge(G) 1 &amp;frac12; inch needles x2 (for performing FNAB)</td><td></td><td></td><td></td></tr><tr><td>25G 1 &amp;frac12; inch needle</td><td></td><td></td><td>for injecting local anesthetic</td></tr><tr><td>FNA syringe holder (&quot;gun&quot;)</td><td></td><td></td><td></td></tr><tr><td>bandage</td><td></td><td></td><td></td></tr><tr><td>vial of glutaraldehyde</td><td></td><td></td><td>for electron microscopy</td></tr><tr><td>biopsy container of 10% formalin</td><td></td><td></td><td></td></tr></tbody></table>

performing and processing fna of anterior fat pad for amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

performing-and-processing-fna-of-anterior-fat-pad-for-amyloid

Nanyang Technological University

Research

JoVE Journal

Medicine

55.0K Views.  Medical College of Wisconsin. Vetkussentje aspiratie is een voorkeur, minimaal invasieve, en lage kosten aanpak in vergelijking met andere methoden om amyloïd te detecteren voor de diagnose van systemische amyloïdose. Deze video artikel wordt een procedurele schets voor het uitvoeren van dikke pad aspiratie met de juiste verwerking van het monster voor de optimale diagnostische resultaat.

Video: Performing and Processing FNA of Anterior Fat Pad for Amyloid

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Biochemistry

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Neuroscience

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfr&#945;-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

The replacement of healthy muscle tissue with intramuscular fat is a prominent feature of human diseases and conditions. This protocol outlines how to visualize, image, and quantify intramuscular fat, allowing the rigorous study of the mechanisms underlying intramuscular fat formation.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build ...

Developmental Biology

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find preventive and curative measures. The most effective way to achieve this is through direct examination of diseased and healthy brain tissue. The authors present a protocol to obtain, process, characterize and store good quality brain tissue donated by individuals registered in an antemortem brain donation program. The donation program includes a face-to-face empathic approach to people, a collection of complementary clinical, biological, social and lifestyle information and serial multi-dimensional assessments over time to track individual trajectories of normal aging and cognitive decline. Since many neurological diseases are asymmetrical, our brain bank offers a unique protocol for slicing fresh specimens. Brain sections of both hemispheres are alternately frozen (at -80 &#176;C) or fixed in formalin; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. With this approach, a complete histological characterization of all frozen material can be obtained, and omics studies can be performed on histologically well-defined tissues from both hemispheres thus offering a more complete assessment of neurodegenerative disease mechanisms. Correct and definite diagnosis of these diseases can only be achieved by combining the clinical syndrome with the neuropathological evaluation, which often adds important etiological clues necessary to interpret the pathogenesis. This method can be time consuming, expensive and limited as it only covers a limited geographical area. Regardless of its limitations, the high degree of characterization it provides can be rewarding. Our ultimate goal is to establish the first Italian Brain Bank, all the while emphasizing the importance of neuropathologically verified epidemiological studies.

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Performing and Processing FNA of Anterior Fat Pad for Amyloid

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