Profile analysis of hepatic porcine and murine brain tissue slices obtained with a vibratome

Mattei, Giorgio; Cristiani, Irene; Magliaro, Chiara; Ahluwalia, Arti

doi:10.7717/peerj.932

Cited by 5 publications

(7 citation statements)

References 30 publications

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“…The latter was then cut into 1 mm-thick coronal slices using a Leica VT1200S vibratome (Leica Microsystems, Nussloch, Germany) with a stainless steel razor blade (Gillette, Milan, Italy). The cut settings were: blade angle, 18°; sectioning speed, 0.2 mm/s; and oscillating amplitude, 1.5 mm (Mattei et al, 2015 ). Each hydrogel-embedded slice was placed in a 50 mL Falcon tube at 37°C with 20 mL of CLARITY clearing solution, composed of 200 mM Boric Acid (Farmitalia Carlo Erba spa, Italy) and 4% Sodium Dodecyl Sulphate (SDS, Sigma-Aldrich) (Chung et al, 2013 ).…”

Section: Methodsmentioning

confidence: 99%

Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue

et al. 2016

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Tissue clarification has been recently proposed to allow deep tissue imaging without light scattering. The clarification parameters are somewhat arbitrary and dependent on tissue type, source and dimension: every laboratory has its own protocol, but a quantitative approach to determine the optimum clearing time is still lacking. Since the use of transgenic mouse lines that express fluorescent proteins to visualize specific cell populations is widespread, a quantitative approach to determine the optimum clearing time for genetically labeled neurons from thick murine brain slices using CLARITY2 is described. In particular, as the main objective of the delipidation treatment is to clarify tissues, while limiting loss of fluorescent signal, the "goodness" of clarification was evaluated by considering the bulk tissue clarification index (BTCi) and the fraction of the fluorescent marker retained in the slice as easily quantifiable macroscale parameters. Here we describe the approach, illustrating an example of how it can be used to determine the optimum clearing time for 1 mm-thick cerebellar slice from transgenic L7GFP mice, in which Purkinje neurons express the GFP (green fluorescent protein) tag. To validate the method, we evaluated confocal stacks of our samples using standard image processing indices (i.e., the mean pixel intensity of neurons and the contrast-to -noise ratio) as figures of merit for image quality. The results show that detergent-based delipidation for more than 5 days does not increase tissue clarity but the fraction of GFP in the tissue continues to diminish. The optimum clearing time for 1 mm-thick slices was thus identified as 5 days, which is the best compromise between the increase in light penetration depth due to removal of lipids and a decrease in fluorescent signal as a consequence of protein loss: further clearing does not improve tissue transparency, but only leads to more protein removal or degradation. The rigorous quantitative approach described can be generalized to any clarification method to identify the moment when the clearing process should be terminated to avoid useless protein loss.

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Section: Methodsmentioning

confidence: 99%

Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue

et al. 2016

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“…We developed a new protocol that allowed us to efficiently cut mouse spleens in intact slices and to maintain these alive and responsive for at least 48 h, making them suitable for functional assays. The protocol, that is a modification of protocols developed to obtain precision-cut slices of mouse brain, liver and lung (9)(10)(11), is based on the sequential following steps: (1) spleen inclusion into agarose blocks; (2) precision-cut using a vibrating microtome; and (3) 48-h culture of spleen slices. The protocol developed for the preparation of organotypic cultures of mouse spleens has turned out to be a valuable tool to (i) prepare spleen slices with a sufficient degree of tissue integrity; and (ii) maintain this complex tissue in culture for days, in order to be used for functional assays.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays

et al. 2020

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By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.

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“…12 Commercially available slicers as well as vibratomes can be used, whereby the latter are preferred for slicing of fastidious tissues like the brain, heart, lymph nodes, or retina. [29][30][31] Depending on the tissue, suitable slicing buffers and incubation media need to be selected, as some tissues have a lower tolerance for variations in pH and/or oxygen tension. Cell-dense tissues like the kidney or liver usually require an oxygenated, pHstable environment during slicing and incubation to assure viability, whereas lung tissue needs to be stabilized prior to slicing.…”

Section: Precision-cut Tissue Slicesmentioning

confidence: 99%

“…34 PCTS suitability for established endpoint analyses has been demon-strated in numerous studies. Cytokine/chemokine release, 35 enzyme activation/inhibition, 36 ion channel activity analyses, 37 mRNA/protein expression, 38 pathohistology, 29 and immunohistochemistry, 39 to only name a few-the list of possible endpoints is equally as comprehensive as for in vitro/in vivo methods.…”

Section: Pcts Advantages and Limitations For Cwa Researchmentioning

confidence: 99%

“…As mentioned above, to ensure that tissue slices are uniform in size and thickness, a prerequisite is an adequate slicing device 12 . Commercially available slicers as well as vibratomes can be used, whereby the latter are preferred for slicing of fastidious tissues like the brain, heart, lymph nodes, or retina 29–31 . Depending on the tissue, suitable slicing buffers and incubation media need to be selected, as some tissues have a lower tolerance for variations in pH and/or oxygen tension.…”

Section: Precision‐cut Tissue Slicesmentioning

confidence: 99%

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Chemical warfare agent research in precision‐cut tissue slices—a useful alternative approach

Herbert

Laskin

Gow

et al. 2020

Annals of the New York Academy of Sciences

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The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo models, progress to elucidate mechanisms of toxicity and to develop effective therapies, decontamination procedures, and general countermeasures is still limited. Novel scientific approaches to address these questions are needed to expand perspectives on existing knowledge and gain new insights. To achieve this, the use of ex vivo techniques like precision-cut tissue slices (PCTSs) can be a valuable approach. Existing studies employing this economical and relatively easy to implement method show model suitability and comparability with the use of in vitro and in vivo models. In this article, we review research on CWAs in PCTSs to illustrate the advantages of the approach and to promote future applications.

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Profile analysis of hepatic porcine and murine brain tissue slices obtained with a vibratome

Cited by 5 publications

References 30 publications

Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue

Clarifying CLARITY: Quantitative Optimization of the Diffusion Based Delipidation Protocol for Genetically Labeled Tissue

Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays

Chemical warfare agent research in precision‐cut tissue slices—a useful alternative approach

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