A simple and economical method for the manual construction of well-aligned tissue arrays

Tissue array technology has allowed a substantial progression of studies correlating molecular and immunohistochemical findings with clinico-pathological information. Array construction presents technical difficulties and tissue arrayers are expensive, particularly for small and medium sized laboratories. We describe a simple manual method for producing well-aligned tissue arrays using a hand-made paper mold which can successfully perform immunohistochemical staining. All 200 tissue samples were collected and constructed into blocks by the paper mold. The tissue arrays were smoothly sectioned using a standard microtome and performed for a panel of immunohistochemical study with satisfactory results. This alterative method for building custom arrays could be applied in any laboratory and is both simple and economical.     

A simple and economical method for the manual construction of frozen tissue arrays

Tsao S‐C, Wu C‐C, Wen C‐H, Huang Y‐C, Chai C‐Y. A simple and economical method for the manual construction of frozen tissue arrays. APMIS 2010; 118: 739–43. Tissue microarray has been developed to enable multiple cores of tissue in one or more new paraffin blocks. Currently, almost all tissue microarrays are made by coring cylindrical tissues from formalin‐fixed and paraffin‐embedded tissues. The disadvantages of formalin‐fixed and paraffin‐embedded tissues include the poor preservation of antigenicity of certain proteins and mRNA degradation induced by the fixation and embedding process. However, frozen tissue array construction presents technical difficulties, and tissue array devices are expensive, particularly for small‐ and medium‐sized laboratories. We describe a simple manual method for producing well‐aligned tissue arrays by a capsule freeze method that allows us to successfully perform hematoxylin–eosin and immunohistochemical stain. All 120 tissue samples were collected and constructed into blocks by this capsule freeze method. The capsules were not affected during the sectioning process, and the capsule material always disappeared during the aqueous steps of the stain processing. The frozen tissue arrays were smoothly sectioned without the use of a tape transfer system and immunohistochemical study was performed with satisfactory results. This alternative method can be applied in any laboratory, and is both simple and economical.     

A simple method for the construction of small format tissue arrays

Tissue arrays can evaluate molecular targets in high numbers of samples in parallel. Array construction presents technical difficulties and tissue arrayers are expensive, particularly for small and medium sized laboratories. This report describes a method for the construction of 36 sample arrays using widely available materials. A blunted 16 gauge needle for bone marrow aspiration was used to extract paraffin wax cylinders and manually define a 6 x 6 matrix on a blank paraffin wax block. Tissue cores from 36 paraffin wax embedded premalignant lesions and invasive cervical carcinomas were injected into the matrix using a 14 gauge needle. This tissue array was sectioned using a standard microtome and used for the immunodetection of CD44 variant 9 and interleukin 18 with satisfactory results. This method can be applied in any laboratory, without the need of specialised equipment, offering a good alternative for the wider application of tissue arrays.     

Microfabricated fountain pens for high-density DNA arrays

We used photolithographic microfabrication techniques to create very small stainless steel fountain pens that were installed in place of conventional pens on a microarray spotter. Because of the small feature size produced by the microfabricated pens, we were able to print arrays with up to 25,000 spots/cm2, significantly higher than can be achieved by other deposition methods. This feature density is sufficiently large that a standard microscope slide can contain multiple replicates of every gene in a complex organism such as a mouse or human. We tested carryover during array printing with dye solution, labeled DNA, and hybridized DNA, and we found it to be indistinguishable from background. Hybridization also showed good sequence specificity to printed oligonucleotides. In addition to improved slide capacity, the microfabrication process offers the possibility of low-cost mass-produced pens and the flexibility to include novel pen features that cannot be machined with conventional techniques.     

Polytrodes: high-density silicon electrode arrays for large-scale multiunit recording

We developed a variety of 54-channel high-density silicon electrode arrays (polytrodes) designed to record from large numbers of neurons spanning millimeters of brain. In cat visual cortex, it was possible to make simultaneous recordings from >100 well-isolated neurons. Using standard clustering methods, polytrodes provide a quality of single-unit isolation that surpasses that attainable with tetrodes. Guidelines for successful in vivo recording and precise electrode positioning are described. We also describe a high-bandwidth continuous data-acquisition system designed specifically for polytrodes and an automated impedance meter for testing polytrode site integrity. Despite having smaller interconnect pitches than earlier silicon-based electrodes of this type, these polytrodes have negligible channel crosstalk, comparable reliability, and low site impedances and are capable of making high-fidelity multiunit recordings with minimal tissue damage. The relatively benign nature of planar electrode arrays is evident both histologically and in experiments where the polytrode was repeatedly advanced and retracted hundreds of microns over periods of many hours. It was possible to maintain stable recordings from active neurons adjacent to the polytrode without change in their absolute positions, neurophysiological or receptive field properties.     

The construction of high-density paraffin tissue microarrays with 0.43-mm-diameter paraffin tissue core biopsies is technically feasible

Paraffin tissue microarrays (PTMAs) are blocks of paraffin containing up to 1,000 paraffin tissue core biopsies (PTCBs). The growing number of publications in recent years bears eloquent witness to the advantages of these PTMAs in high-throughput molecular profiling of tumor specimens. In order to conserve the often minute quantities of available tumor tissue with precisely recorded follow-up data and to store the greatest possible number of PTCBs in one block, researchers often try to reduce PTCBs to the smallest possible diameter. Until now, the smallest feasible diameter for PTCBs was 0.6 mm. Experiments with diameters below 0.6 mm have failed due to the instability of the paraffin tissue punch. The process described allows the construction of PTMAs with PTCBs only 0.43 mm in diameter utilizing simple, inexpensive, self-made paraffin tissue punches and predrilled recipient blocks.     

A quick and easy method for the staining of reticulocytes.

This report presents a simplification of the conventional method for the staining of reticulocytes that is easier, faster and requires no extraneous equipment. Blood is applied directly to a spot of dried stain on a microscope slide. An identical slide is placed over the first and the blood and stain are mixed for about a minute. The slides are held apart by paper labels at each end. The slides are then slid apart to produce a smear on each slide.     

Flexible use of high-density oligonucleotide arrays for single-nucleotide polymorphism discovery and validation

A method for identifying and validating single nucleotide polymorphisms (SNPs) with high-density oligonucleotide arrays without the need for locus-specific polymerase chain reactions (PCR) is described in this report. Genomic DNAs were divided into subsets with complexity of ~10 Mb by restriction enzyme digestion and gel-based fragment size resolution, ligated to a common adaptor, and amplified with one primer in a single PCR reaction. As a demonstration of this approach, a total of 124 SNPs were located in 190 kb of genomic sequences distributed across the entire human genome by hybridizing to high-density variant detection arrays (VDA). A set of independent validation experiments was conducted for these SNPs employing bead-based affinity selection followed by hybridization of the affinity-selected SNP-containing fragments to the same VDA that was used to identify the SNPs. A total of 98.7% (74/75) of these SNPs were confirmed using both DNA dideoxynucleotide sequencing and the VDA methodologies. With flexible sample preparation, high-density oligonucleotide arrays can be tailored for even larger scale genome-wide SNP discovery as well as validation.     

An inverse method to design RF coil arrays optimized for SENSE imaging

A new method to design MRI RF coils that are optimized for SENSE (sensitivity encoding) imaging is introduced. In this approach, the inverse problem was solved where the surface current density distribution on a coil former was calculated to maximize the SNR(sense) within a volume of interest (VOI). For that purpose, an analytic relationship was formulated between the SNR(sense) and surface current density on the coil former. The SNR at pixel rho in a SENSE-MR image, SNR(sense,rho), is inversely proportional to the g-factor: therefore, the g-factor was formulated in terms of the B1 distribution of the coils. Then, by specifying the geometry of the desired coil former and using a finite element mesh (FEM), the surface current distribution was calculated to maximize the SNR(sense), by minimizing (1/SNR(sense)) in the VOI using a least squares procedure. A simple two-coil array was designed and built to test the method and phantom images were collected. The results show that the new coil design method yielded better uniformity and SNR in SENSE images compared to those of standard coils.     

Amplification of tissue by construction of tissue microarrays.

Tissue microarrays are a method of relocating tissue from conventional histologic paraffin blocks in a manner that tissue from multiple patients or blocks can be seen on the same slide. This is done by using a needle to biopsy a standard histologic section and placing the core into an array on a recipient paraffin block. This technique allows maximization of tissue resources by analysis of small core biopsies of blocks, rather than complete sections. Using this technology, a carefully planned array can be constructed using cases from pathology tissue block archives, and a 20-year survival analysis can be done on a cohort of 600 or more patients using only a few microliters of antibody in a single experiment. Furthermore, this cohort can be analyzed thousands of times with different reagents as a result of judicious sectioning of the array block. This review describes this process and discusses the issues of representative sampling in heterogeneous lesions, the issue of antigen preservation, and some technical strategies and methods of array construction. In summary, this technique can provide a highly efficient, high-throughput mechanism for evaluation of protein expression in large cohorts. It has the potential for allowing validation of new genes at a speed comparable to the rapid rate of gene discovery afforded by DNA microarrays.     

An in-house ELISA method for quantification of circulating tissue factor

Tissue Factor (TF) is a low molecular weight transmembrane glycoprotein that initiates the clotting protease cascade. It is considered to be the principal regulator of the extrinsic coagulation pathway, hemostasis and thrombosis, as well as inflammation and cellular immune response. An in-house two-step direct sandwich ELISA (enzyme-linked immunosorbent assay) for immunological quantification of plasma TF was successfully developed. The assay employed a monoclonal antibody against human TF (1:400 dilution; 1250 ng/ml) and peroxidase-conjugated anti-TF IgG (1:1000 dilution; 2000 ng/ml) as capture and detecting antibodies respectively, whilst tetramethylbenzidine/H2O2 were utilized as substrates. Titration curves of recombinant TF were linear within 10 to 4000 pg/ml, with a detection limit of 36.31 pg/ml. It demonstrated low intra- (2.50 - 9.23 CV%) and inter-assays (5.65 - 13.57 CV%) variability, as well as satisfactory analytical recovery (91.55 - 103.95%) and good parallelism. The assay developed was intended to be applied for measurement of plasma TF levels in patients with thrombotic disorders.     

Rapid method for detection of Coxiella burnetii antibodies using high-density particle agglutination.

A high-density particle agglutination test, using erythrocyte-sensitizing substance from phase II Coxiella burnetii adsorbed to high-density composite particles, was developed for rapid serodiagnosis of Q fever. The test was compared with the microimmunofluorescence test for sensitivity and specificity by using 3,036 human serum samples collected in Gifu Prefecture, Japan. An excellent agreement was found between the two tests for the acute-phase group and paired serum samples, but some discordant results were observed in the single-sample group. The sensitivity and specificity of the high-density particle agglutination test were both 100% in the former group and 81.6 and 99.9%, respectively, in the latter group. The test is a very promising tool for routine serodiagnosis of Q fever because of its simplicity, sensitivity, and specificity.     

Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays

Large scale human genetic studies require technologies for generating millions of genotypes with relative ease but also at a reasonable cost and with high accuracy. We describe a highly parallel method for genotyping single nucleotide polymorphisms (SNPs), using generic high-density oligonucleotide arrays that contain thousands of preselected 20-mer oligonucleotide tags. First, marker-specific primers are used in PCR amplifications of genomic regions containing SNPs. Second, the amplification products are used as templates in single base extension (SBE) reactions using chimeric primers with 3' complementarity to the specific SNP loci and 5' complementarity to specific probes, or tags, synthesized on the array. The SBE primers, terminating one base before the polymorphic site, are extended in the presence of labeled dideoxy NTPs, using a different label for each of the two SNP alleles, and hybridized to the tag array. Third, genotypes are deduced from the fluorescence intensity ratio of the two colors. This approach takes advantage of multiplexed sample preparation, hybridization, and analysis at each stage. We illustrate and test this method by genotyping 44 individuals for 142 human SNPs identified previously in 62 candidate hypertension genes. Because the hybridization results are quantitative, this method can also be used for allele-frequency estimation in pooled DNA samples.     

Quick and easy microfabrication of T-shaped cantilevers to generate arrays of microtissues

Over the past decade, a major effort was made to miniaturize engineered tissues, as to further improve the throughput of such approach. Most existing methods for generating microtissues thus rely on T-shaped cantilevers made by soft lithography and based on the use of negative SU-8 photoresist. However, photopatterning T-shaped microstructures with these negative photoresists is fastidious and time-consuming. Here we introduce a novel method to quickly generate T-shaped cantilevers dedicated to generation of cellular microtissues, based on the use of positive photoresist. With only two layers of photoresist and one photomask, we were able to fabricate arrays of microwells in less than 3 h, each containing two T-shaped cantilevers presenting either a rectangular or a circular geometry. As a proof of concept, these arrays were then replicated in poly(dimethylsiloxane) and microtissues composed of NIH 3T3 fibroblasts encapsulated in collagen I were generated, while the two cantilevers simultaneously constrain and report forces generated by the microtissues. Immunostainings showed longitudinally aligned and elongated fibroblasts over the whole microtissue after 8 days of culture. The method described here opens the potential to quick prototyping platforms for high-throughput, low-volume screening applications.