Complementary Techniques

Esposito, Giovanni

doi:10.1007/978-0-387-39978-2_6

Cited by 45 publications

(12 citation statements)

References 30 publications

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“…This may help to identify new components that influence in vivo cell behaviour such as migration. Furthermore, if combined with laser capture microdissection and transcardial fixation, it may be possible to dissect tissue of which the in vivo history is known and which can be used for further analyses such as mass spectrometry 16 , microarray analysis 17 or genomic sequencing 18 . Primary invasive lobular carcinoma tumours are a heterogeneous population of cells that includes cancer cells and a variety of immune cells such as T cells and macrophages 19 .…”

Section: Discussionmentioning

confidence: 99%

In vivo imaging and histochemistry are combined in the cryosection labelling and intravital microscopy technique

2013

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Section: Discussionmentioning

confidence: 99%

In vivo imaging and histochemistry are combined in the cryosection labelling and intravital microscopy technique

2013

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“…Histopathology has historically utilized laser capture microdissection to isolate specific biomolecules from microscale regions of interest in thinly sliced (<10um thick) sections (5,6). Despite recent improvements, this technique is limited by the need for expensive equipment, low throughput/long processing times, experienced operators.…”

Section: Introductionmentioning

confidence: 99%

Vital Ex Vivo Tissue Labeling and Pathology-Guided Micropunching to Characterize Cellular Heterogeneity in the Tissue Microenvironment

et al. 2018

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Cellular heterogeneity within the tissue microenvironment may underlie chemotherapeutic resistance and response, enabling tumor evolution but is difficult to characterize. Here we present an approach, dubbed pathology guided micropunching (PGM) that enables the identification and further characterization of heterogeneous foci identified in viable human and animal model tissue slices. This technique consists of a live-cell tissue labeling approach using fluorescent antibodies/small molecules to identify heterogeneous foci, including immune infiltrates and cells high in reactive oxygen species in viable tissues. This is coupled with a micropunch technique to isolate cells from these heterogeneous foci for downstream molecular or vital functional analysis. Micropunches targeted to epithelial or stromal fibroblast foci in human prostate tissue show 6–12 fold enrichment in transcripts specific for EpCam/Cytokeratin 8 and Vimentin/alpha-Smooth Muscle Actin/Integrin 1-alpha, respectively. Transcriptional enrichment efficiency is in-line with epithelial and stromal laser capture microdissection samples isolated from human prostate. Micropunched foci show a peripheral loss of cellular viability, but centrally localized cells retained viability before and after dissociation and grew out in culture. Application of this technique can advance our understanding of cellular heterogeneity across the tissue microenvironment leading to tumor evolution and other states of health and disease.

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“…This necessitates a clear separation between the tissue section of interest and surrounding tissues sections(4). With the current advancements in molecular profiling technology, there has been an increasing need to assess tissue at the cellular level.…”

Section: Introductionmentioning

confidence: 99%

“…First, LCM is an expensive and time consuming process. Additionally, due to the unstable nature of RNA, it is often challenging to obtain high quality RNA from LCM samples(4). Due to the disadvantages of LCM, new advances in microdissection technology are still needed to make it more accessible to a greater number of researchers in an affordable and time sensitive manner.…”

Section: Introductionmentioning

confidence: 99%

A Novel Microdissection Approach to Recovering <em>Mycobacterium tuberculosis </em>Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Hudock¹,

Kaushal

2014

JoVE

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SHORT ABSTRACT Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, has recently now available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas derived from experimentally infected nonhuman primates. LONG ABSTRACT Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser Capture Microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section(1). The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limit its efficient use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mil bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hemotoxylin and eosin stained (H and E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage of the system is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) is a major infectious disease killer of humanity worldwide and results from infection with Mtb. In a majority of individuals exposed to the aerosols of Mtb, the infection is latently limited but in at least 10 million people annually, it results in active TB disease(2). During latent infection, Mtb is contained within pathological lung lesions known as the granuloma. Hence, it has been argued that the outcome of Mtb infections is decided at the level of the granuloma(3).

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Complementary Techniques

Cited by 45 publications

References 30 publications

In vivo imaging and histochemistry are combined in the cryosection labelling and intravital microscopy technique

In vivo imaging and histochemistry are combined in the cryosection labelling and intravital microscopy technique

Vital Ex Vivo Tissue Labeling and Pathology-Guided Micropunching to Characterize Cellular Heterogeneity in the Tissue Microenvironment

A Novel Microdissection Approach to Recovering <em>Mycobacterium tuberculosis </em>Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

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