An improved method for embedding brain tissue in albumin-gelatin.

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose, and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 micron. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

Rapid removal of acid from glycol methacrylate for improved histological embedding.

A method for the rapid and complete removal of methacrylic acid from commercial samples of glycol methacrylate is presented. It entails conversion of the acid to an insoluble N-acylurea by treatment with an equivalent amount of N,N'-dicyclohexylcarbodiimide. Sections of tissue embedded in polymer prepared from the purified monomer can be stained with basic dyes without simultaneously staining the polymer.     

A polymethyl methacrylate method for large specimens of mineralized bone with implants

A simple modified polymethyl methacrylate method is described for large mineralized bone specimens with implants and bioactive materials which produces consistently good histological preservation of the interface between bone and implant. Human femoral heads, whole rabbit condyles and canine tibias and femurs containing implants consisting of hydroxyapatite, smooth polyethylene, porous polyethylene and carbon were dehydrated in ascending grades of ethanol and cleared with xylene on an automated tissue processor which alternated vacuum and pressure for 22 hr. Infiltration was done with washed polymethyl methacrylate at 4 C under vacuum for 13 days. Polymerization was carried out in wide-mouth glass jars at 38 C for 36 hr so that the total processing time was less than 20 days. The only important modification was in the polymethyl methacrylate, which had less plasticizer than usual in order to give a harder block. This enabled production of 4 micron sections with good preservation of mineralized and cellular areas for the study of metabolic bone diseases, morphometry, fluorochrome labelling and interface analysis with the implant in situ.     

An improved flat embedding technique for immunoelectron microscopy

Immunocytochemical staining has been widely used for localizing various hormonal antigens, protein markers and putative neurotransmitters in tissues. Immunostained sections can be examined light microscopically and specific areas selected for electron microscopic study.     

An improved method for the fluorometric determination of tissue catecholamines

Catecholamines extracted from tissues are readily measured by fluorescence following trihydroxyindole derivatization. A complicating factor however, has been the variably high background fluorescence levels obtained which adversely affect the precision of the assay. Employing EDTA is shown to reduce the high background fluorescence to a consistently low level, and to improve the accuracy of the trihydroxyindole method. It is suggested that high background fluorescence readings obtained by previous workers were due to metal ions extracted from tissue and carried over into the derivatization procedure.Measurement of tissue catecholamines by fluorescent methods is improved by the addition of EDTA which gives a consistently low background with a concommitant increase in accuracy.     

An improved method for tissue long-chain acyl-CoA extraction and analysis

We report an extensively modified method for the extraction, solid-phase purification, and HPLC analysis of long-chain acyl-CoAs from tissues. Tissue samples were homogenized in a glass homogenizer in KH2PO4 buffer (100 mM, pH 4.9) and again after the addition of 2-propanol. Acyl-CoAs were then extracted from the homogenate with acetonitrile (ACN). The acyl-CoAs in the extract were bound to an oligonucleotide purification column and eluted using 2-propanol. This eluent was concentrated and then loaded onto a C-18 column and eluted using a binary gradient system in which solvent A was KH2PO4 (75 mM, pH 4.9) and solvent B was ACN containing 600 mM glacial acetic acid. Initial flow rate was 0.5 or 0.25 ml/min depending upon the tissue used. The HPLC eluent was monitoring at 260 nm. Our modifications increased the recovery of the extraction procedure to 70-80%, depending upon tissue, with high reproducibility and significantly improved separation of the most common unsaturated and saturated acyl-CoAs. We also report, for the first time, the mass (nanomoles per gram wet weight) of the most common polyunsaturated acyl-CoAs in rat heart, kidney, and muscle tissues. The modifications and high recovery permit the use of tissue samples of less than 100 mg, making this method useful for the analysis of small tissue amounts associated with mice.     

An improved method for isolating RNA from porcine adipose tissue.

In the present study, we describe a method that we developed to isolate total RNA from porcine adipose tissue. This method entails homogenizing porcine adipose tissue in 10 ml of 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% Sarcosyl, 0.1 M beta-mercaptoethanol, pH 7.0, and then performing two CHCl3 extractions to remove lipid before following the procedure described by P. Chomczynski and N. Sacchi (1987, Anal. Biochem. 162, 156-159). This modification improved the yield of RNA approximately threefold (yield was 88 +/- 7 micrograms total RNA/g of tissue) without affecting RNA quality.     

Glycol methacrylate embedding in histotechnology: the hematoxylin-eosin stain as a method for assessing the stability of glycol methacrylate sections

Glycol methacrylate (GMA) samples containing inhibitor in the range of 200-300 ppm were included in a standard embedding mixture. The pH of the GMA samples was measured as a 10% solution of the sample in distilled water. The acidity of GMA due to methacrylic acid causes background staining of sections after basic dyes. The concentration of GMA and the amount of impurities such as methacrylic acid (MA) and ethylene glycol dimethacrylate (EDMA) were measured by gas chromatography. Distinct variations in purity were found among five samples of GMA. Sections derived from GMA samples containing more than 2% EDMA showed few, if any, minifolds after staining with hematoxylin and eosin and were more stable in alcoholic and basic solutions; sections from purer GMA showed minifolds and were less stable. Addition of crosslinkers, EDMA or triethylene glycol dimethacrylate (TEDMA) prevented these artifacts. Crosslinkers clearly influence dimensional changes in sections. Addition of crosslinkers to GMA samples containing minimal amounts of MA improved the results. The possibility of obtaining a high quality GMA embedding medium is discussed.     

Fabrication of a capillary immunosensor in polymethyl methacrylate.

A method for fabricating capillary-based immunosensors in a coupon milled from an inexpensive, commodity plastic (PMMA, plexiglass) is demonstrated. The key feature of the technique is the use of sol-gel technology to deposit a glass-like (Si [bond] OH) film on surfaces of the plastic capillary channels to facilitate antibody immobilization. The utility of this method was demonstrated in the context of continuous flow displacement immunosensors for the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). These sensors exhibited sensitivity to low microg/l RDX concentrations and peak-to-peak signal variations that were generally less than 10% for multiple injections at a single RDX concentration. The useful lifetime of the coupons in these experiments was greater than 10 h even after multiple exposures to high (1000 microg/l) RDX levels. This sensor platform has the physical characteristics needed for a portable field instrument: small, light-weight, and rugged.     

An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.