scholarly journals Optimisation and validation of hydrogel-based brain tissue clearing shows uniform expansion across anatomical regions and spatial scales

2018 ◽  
Author(s):  
Adam L. Tyson ◽  
Ayesha Akhtar ◽  
Laura C. Andreae
Keyword(s):  
Brain Tissue ◽  
Quantitative Assessment ◽  
Spatial Scales ◽  
Fixed Tissue ◽  
Optical Clearing ◽  
Tissue Clearing ◽  
Tissue Morphology ◽  
Conventional Methods ◽  
Uniform Expansion

ABSTRACTImaging of fixed tissue is routine in experimental neuroscience, but is limited by the depth of tissue that can be imaged using conventional methods. Optical clearing of brain tissue using hydrogel-based methods (e.g. CLARITY) allows imaging of large volumes of tissue and is rapidly becoming commonplace in the field. However, these methods suffer from a lack of standardised protocols and validation of the effect they have upon tissue morphology. We present a simple and reliable protocol for tissue clearing along with a quantitative assessment of the effect of tissue clearing upon morphology. Tissue clearing caused tissue swelling (compared to conventional methods), but this swelling was shown to be similar across spatial scales and the variation was within limits acceptable to the field. The results of many studies rely upon an assumption of uniformity in tissue swelling, and by demonstrating this quantitatively, research using these methods can be interpreted more reliably.

scholarly journals A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

10.3791/61821 ◽  
2020 ◽  
Author(s):  
Kristin N. Kirchner ◽  
Hailong Li ◽  
Adam R. Denton ◽  
Steven B. Harrod ◽  
Charles F. Mactutus ◽  
...  
Keyword(s):  
Rat Brain ◽  
Brain Tissue ◽  
Tissue Clearing ◽  
Clearing Method ◽  
Rat Brain Tissue

scholarly journals Mechanical Compression of Coverslipped Tissue Sections During Heat-induced Antigen Retrieval Prevents Section Detachment and Preserves Tissue Morphology

2019 ◽  
Vol 67 (6) ◽  
pp. 441-452 ◽  
Author(s):  
Andreas H. Eckhard ◽  
Jennifer T. O’Malley ◽  
Joseph B. Nadol ◽  
Joe C. Adams
Keyword(s):  
Antigen Retrieval ◽  
Microscope Slide ◽  
Major Drawback ◽  
Fixed Tissue ◽  
Simple Method ◽  
Tissue Sections ◽  
Tissue Morphology ◽  
Glass Slides ◽  
Tissue Antigens ◽  
Temporal Bones

Heat-induced antigen retrieval (HIAR) is routinely employed on aldehyde-fixed tissue sections to enhance the reactivity of antibodies that exhibit weak or no specific interactions with tissue antigens when applied in conventional immunohistochemical protocols. A major drawback of HIAR protocols is, however, the heat-induced detachment of sections from the microscope slide with resultant impaired tissue morphology or loss of the section. We developed a method in which tissue sections mounted on glass slides are temporally coverslipped, and a clamp is used to compress the sections on the microscope slide during HIAR treatment. This “pressurized coverslipping” during HIAR was tested on various formalin-fixed tissues (murine kidneys and temporal bones, human tonsils and temporal bones) that were embedded in paraffin or celloidin. The method reliably kept the sections adherent to the slide, preserved the tissue morphology, and effectively retrieved tissue antigens for improved results in immunohistochemical labeling, even for exceptionally delicate, large, and poorly adhering sections, that is, decalcified human temporal bone sections. In summary, we present a simple method for improved slide adherence and morphological preservation of tissue sections during HIAR treatment that can be combined with all HIAR protocols and that requires only basic lab equipment.

scholarly journals Microwaves: A Revolution in Histoprocessing

2014 ◽  
Vol 15 (2) ◽  
pp. 149-152 ◽  
Author(s):  
N Amrutha
Keyword(s):  
Microwave Processing ◽  
Microwave Method ◽  
Diagnostic Ability ◽  
Chi Square ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Chi Square Test ◽  
Conventional Methods ◽  
Formalin Fixed ◽  
Reliable Diagnostic Method

ABSTRACT Background and Aim Pathologists are under constant pressure for instant and reliable diagnosis. The manual procedures employed in private laboratories and institutional setup for histoprocessing and staining are laborious and intense. Thus, this study aims to evaluate and compare the microwave tissue processing and staining with the conventional methods which are in vogue. Materials and methods Of the formalin fixed tissue biopsies received by our department, 30 specimens were randomly picked and subjected to grossing. Each specimen was cut into equal halves, each half was processed and stained by conventional method while the other by the microwave method. The entire procedure was blinded and evaluated by four observers based on the criteria of Mahesh Babu et al (2011): Cellular clarity, cytoplasmic details, nuclear detail and color intensity. The results were statistically analyzed using Chi square test and kappa. Results The overall time employed for microwave processing was 2 hours and for conventional methods it was 7 hours, while H and E staining by microwave process took 16 minutes and 45 seconds and it took 31 minutes and 20 seconds by the conventional process. The diagnostic ability of microwave method yielded promising results and was less time consuming. Conclusion Microwave processing and staining yielded quicker and better results compared to the routine methods. Therefore, Microwave can serve as a quicker and a reliable diagnostic method for a pathologist. How to cite this article Amrutha N, Patil S, Rao RS. Microwaves: A Revolution in Histoprocessing. J Contemp Dent Pract 2014;15(2):149-152.

scholarly journals Rapid and prodium iodide-compatible optical clearing method for brain tissue based on sugar/sugar-alcohol

2016 ◽  
Vol 21 (8) ◽  
pp. 081203 ◽  
Author(s):  
Tingting Yu ◽  
Yisong Qi ◽  
Jianru Wang ◽  
Wei Feng ◽  
Jianyi Xu ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Sugar Alcohol ◽  
Optical Clearing ◽  
Clearing Method

scholarly journals Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 120 ◽  
Author(s):  
Rene Edgar Condori ◽  
Michael Niezgoda ◽  
Griselda Lopez ◽  
Carmen Acosta Matos ◽  
Elinna Diaz Mateo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dominican Republic ◽  
Real Time ◽  
Brain Tissue ◽  
Border Region ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Genetic Typing ◽  
Formalin Fixed

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing.

scholarly journals hFRUIT: An optimized agent for optical clearing of DiI-stained adult human brain tissue

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sven Hildebrand ◽  
Anna Schueth ◽  
Klaus von Wangenheim ◽  
Christian Mattheyer ◽  
Francesco Pampaloni ◽  
...  
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Human Brain Tissue ◽  
Optical Clearing ◽  
Adult Human Brain ◽  
Adult Human

scholarly journals Ultra-deep through-skull mouse brain imaging via the combination of skull optical clearing and three-photon microscopy

2021 ◽  
Author(s):  
Mubin He ◽  
Dongyu Li ◽  
Zheng Zheng ◽  
Hequn Zhang ◽  
Tianxiang Wu ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Brain Structures ◽  
Brain Vessels ◽  
Intact Mouse ◽  
Optical Clearing ◽  
Cranial Window ◽  
Dual Channel ◽  
Imaging Depth ◽  
Cerebrovascular Imaging

Optical microscopy has enabled in vivo monitoring of brain structures and functions with high spatial resolution. However, the strong optical scattering in turbid brain tissue and skull impedes the observation of microvasculature and neuronal structures at large depth. Herein, we proposed a strategy to overcome the influence induced by the high scattering effect of both skull and brain tissue via the combination of skull optical clearing (SOC) technique and thee-photon fluorescence microscopy (3PM). The Visible-NIR-II compatible Skull Optical Clearing Agents (VNSOCA) we applied reduced the skull scattering and water absorption in long wavelength by refractive index matching and H2O replacement to D2O respectively. 3PM with the excitation in the 1300-nm window reached 1.5 mm cerebrovascular imaging depth in cranial window. Combining the two advanced technologies together, we achieved so far the largest cerebrovascular imaging depth of 1 mm and neuronal imaging depth of >700 μm through intact mouse skull. Dual-channel through-skull imaging of both brain vessels and neurons was also successfully realized, giving an opportunity of non-invasively monitoring the deep brain structures and functions at single-cell level simultaneously.

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