Navigation Links
Innovative Tissue Array Technology for High-Throughput Screening of Gene,,,Expression


Many different tissue types on a convenient single-slide format

Carolyn Thomas
Stratagene

Stratagenes Complete View tissue array slides offer further refinement of tissue array technology through optimized tissue fixation and processing procedures that preserve tissue morphology, antigenicity of proteins, and nucleic acid reactivity. This innovative product line consists of murine (male and female) survey and validation array slides. The Complete View survey array slides represent major organ systems, consisting of approximately 104 cell types mounted in duplicate. The Complete View validation array slides include 12 representative tissues from the survey arrays and are useful to test staining procedures and reagents. We report our results using Complete View array slides for hematoxylin and eosin staining of murine tissue, immunostaining of murine tropomyosin, and labeling RNA of murine salivary gland. These tests confirm that Complete View tissue array slides ensure reliable screening results in a convenient, high-throughput format.

To achieve an understanding of the cellular function of gene products requires localizing gene expression to specific cells and tissues. This task can be accomplished through the use of tissue arrays, a collection of diverse tissue samples mounted on a single slide. Proven successful in examining both normal1,2 and cancerous tissues,3,4 tissue arrays can be treated as a single histological slide during staining, immunohistochemistry, or in situ hybridization. The single-slide format affords a high-throughput method for precisely identifying the specific tissues and cell types that express a gene of interest.

Single-Slide Whole-Animal Surveys

One particularly useful application of tissue arrays is the whole-animal survey, in which all tis sues and cell types in an animal species are screened. Traditional sections for whole-animal surveys require approximately 25 to 40 histological slides for complete screening. With each slide requiring preparation and processing that includes staining, labeling, and analysis, such surveys are so demanding of time, labor, and reagents that they are impractical in most research environments. In contrast, a single Complete View tissue array can contain all the major tissues and cell types of an animal. Hence, tissue array technology makes it possible to screen numerous tissues in a single experiment (Figure 1).

Fig.1

Stratagene has improved tissue array technology by creating the Complete View survey and validation array slides. In this product line, optimized tissue fixation preserves morphological integrity, antigenicity of proteins, and nucleic acid reactivity. On each Complete View array slide, tissues are conveniently configured for easy identification with the Complete View target locator, which positions each tissue within the array by column and row. The availability of two categories of array slides provides further convenience. The Complete View survey array slides represent major organ systems that consist of approximately 104 cell types mounted in duplicate (Table 1). The Complete View validation array slides include representative tissues from the survey arrays and are useful to test staining procedures and reagents.

Slides

Tis sue specimens

Murine Validation Array,
Male C57BL/6J or
Female C57BL/6J

12 unique tissue types represented from the following groups:

  • connective tissue
  • skeletal tissue
  • nervous tissue
  • lymphatic tissue
  • integument
  • alimentary system
  • respiratory system
  • urinary system
  • glandular tissue

Each representative tissue has dual representation per slide.

(24 tissue spots per slide + 1 positive and 1 negative control spot)

Murine Survey Array,
Male C57BL/6J or
Female C57BL/6J

In excess of 40 tissue types representing greater than 100 cell types from the following groups:

  • connective tissue
  • skeletal tissue
  • nervous tissue
  • lymphatic tissue
  • integument
  • alimentary system
  • respiratory system
  • urinary system
  • glandular tissue
  • vascular system
  • reproductive system

Preserved Tissue Integrity

We assessed the morphological integrity of tissues and the antigenicity of proteins by assaying the Complete View array slides in hematoxylin and eosin staining and immunohistochemical analysis, respectively. Figure 2 shows multiple tissues stained within the Complete View male mouse survey array slide. To test antigenicity, we used the Complete View male mouse validation array slide as described (Methods) with antitropomyosin as the primary antibody and goat anti-rabbit FITC as the secondary antibody. The result of this immunological screening assay demonstrates the actin-binding protein within the muscle tissue (Figure 3).

Fig.2

Fig.3

Since many applications of tissue array technology rely on nucleic acid assays, we used the Complete View male mouse validation array slides for an in situ hybridization experiment (Figure 4). One of two slides was treated with RNase prior to hybridization of both slides with a fluorescently labeled poly(dT) probe. Salivary gland tissue on the slide without RNase digestion clearly shows the presence of mRNA . We used standard conditions for slide preparation, prehybridization, and hybridization reactions (Methods).

Fig.4

Conclusions

The Complete View tissue array slide kits permit wide- spread use of tissue array technology. With the single-slide format and convenient configuration of tissues, these innovative array slides simplify screening procedures so specialized expertise in histology is not required. We demonstrated their use to localize expression to specific tissues and cells, and our data confirm that these optimized array slides maintain the morphological integrity of tissues. By immunostaining of the tropomyosin protein and fluorescent labeling of RNA, we verified that the tissues affixed to the Complete View slides retain antigenicity of proteins and reactivity of nucleic acids, respectively. We are expanding this product line to include human fetal validation and survey arrays.

Methods

In Situ Hybridization: Paraffin was removed from the array tissue by treating the slides as described in the product instructions. The two consecutive slides were rehydrated and washed in PBS, immersed in 0.1 M glycine, and washed three times in PBS. Then, the slides were incubated with proteinase K and one was subsequently treated with RNase. To stop the reactions, slides were immersed in 4% paraformaldehyde in PBS followed by a PBS rinse. The slides were then incubated with acetic anhydride in triethanolamine and prehybridized in formamide (50% v/v) in 4X SSC at 37C for 30 minutes, then hybridized with 20 ml of poly d(T)-FITC incubated at 42C for 12 to 18 hours. This incubation was followed by a series of washes with 4X SSC. Each slide was examined with an Olympus BX60 fluorescent microscope.

Immunohistochemistry: Paraffin was removed from the array tissue by treating the slide as described in the product instructions. The slide was rehydrated, then incubated in PBS for 5 minutes and placed in a humidity chamber. The diluted primary antibody was added to the slide and incubated for 30 to 60 minutes at 37C. After washing with PBS, the diluted secondary antibody was added to the slide and incubated for 30 to 60 minutes at 37C. Following two additional PBS washes, a coverglass was placed over the slide, and the slide was examined using an Olympus BX60 fluorescent microscope.


REFERENCES
  1. Battifora, H. (1986) Lab. Invest. 55(2): 244-248.
  2. Battifora, H. and Mehta, P. (1990) Lab. Invest. 63(5): 722-724.
  3. Kononen, J., et al. (1998). Nature Med. 4(7): 844-847.
  4. Bubendorf, L., et al. (1999) Cancer Res. 59: 803-806.

'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Detecting Gastrin-Releasing Peptide Receptor by in situ PCR on Archived Tissue
2. Simple Isolation of RNA from Tissue and Cultured Cells
3. Tissue Specificity for Mutation Parallels Tissue Specificity for Cancer
4. Hands-free Tissue Disruption No Homogenization Needed
5. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
6. RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control
7. Stabilizing RNA in Fresh and Frozen Tissues for RNA Isolation
8. Genomic DNA Preparation from RNAlater Preserved Tissues
9. DNA Isolation From 300 to 450 mg Dried or 600 to 1,200 mg Fresh Leaf Tissue
10. DNA Isolation From 50 to 20 mg of Paraffin-Embedded Tissue
11. Genomic DNA Isolation From 510 mg Marine Invertebrate Tissue
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... ... 27, 2016 , ... Cancer experts from Austria, Hungary, Switzerland, ... a new and helpful biomarker for malignant pleural mesothelioma. Surviving Mesothelioma has just ... now. , Biomarkers are components in the blood, tissue or body fluids ...
(Date:6/27/2016)... --  Ginkgo Bioworks , a leading organism design ... awarded as one of the World Economic Forum,s ... innovative companies. Ginkgo Bioworks is engineering biology to ... in the nutrition, health and consumer goods sectors. ... including Fortune 500 companies to design microbes for ...
(Date:6/23/2016)... ... June 23, 2016 , ... Mosio, ... second eBook, “Clinical Trials Patient Recruitment and Retention Tips.” Partnering with experienced clinical ... eBook by providing practical tips, tools, and strategies for clinical researchers. , “The ...
(Date:6/23/2016)... 2016 Houston Methodist Willowbrook Hospital has ... Association to serve as their official health care ... Willowbrook will provide sponsorship support, athletic training services, ... coaches, volunteers, athletes and families. "We ... Association and to bring Houston Methodist quality services ...
Breaking Biology Technology:
(Date:6/15/2016)... , June 15, 2016 ... market report titled "Gesture Recognition Market by Application Market - Global ... 2016 - 2024". According to the report, the  global ... billion in 2015 and is estimated to grow ... 48.56 billion by 2024.  Increasing application ...
(Date:6/7/2016)... , June 7, 2016  Syngrafii Inc. ... a business relationship that includes integrating Syngrafii,s patented ... branch project. This collaboration will result in greater ... the credit union, while maintaining existing document workflow ... http://photos.prnewswire.com/prnh/20160606/375871LOGO ...
(Date:6/2/2016)... 2016   The Weather Company , an IBM Business ... industry-first capability in which consumers will be able to interact ... questions via voice or text and receive relevant information about ... Marketers have long sought an advertising solution that can ... personal, relevant and valuable; and can scale across millions of ...
Breaking Biology News(10 mins):