Techniques for Studying Adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are frozen rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl₂ (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl₂ (2.5%). When procedures described above for immature adipose depots are combined with esterase fining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

Techniques for studying adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl2 (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl2 (2.5%). When procedures described above for immature adipose depots are combined with esterase staining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

A Portable Dry-Ice Storage Chamber for Enhanced Preservation of Enzymes in Frozen Tissue Specimens

To meet the histochemical needs for enzyme procedures in a laboratory not equipped with cold storage facilities giving -75 C or below, the following procedures were adopted and found technically and economically satisfactory. During sectioning interruptions, the mounted tissue block, in the cryostat, is covered with aluminum foil to prevent surface drying; after cutting, the specimens still mounted on their carriers are individually wrapped in aluminum foil and placed in polyethylene bags. These are then placed in the bottom of a 15 × 34 cm Dewar flask, and the flask filled to capacity with approximately 1.9 kg of dry ice.     

A Comparison of Techniques Useful for Preparing Nematodes for Scanning Electron Microscopy

Second-stage juveniles of Meloidogyne incognita were prepared by several different techniques for scanning electron microscopy (SEM). Sequential fixation in the cold (4-8 C) was superior to rapid fixation at room temperature, glutaraldehyde and glutaraldehyde-formalin were better fixatives than formalin alone, and critical point drying with carbon dioxide or Freon gave similar results that were only slightly better than air drying with Freon. Freeze drying sequentially fixed nematodes from 100% ethanol in liquid propane produced the best preserved specimens with the fewest artifacts. Specimens of various free-living and plant-parasitic nematodes were prepared for SEM by freeze drying. This technique was adequate for most genera but unsatisfactory for a few. Although each genus may require a different procedure for optimum preservation of detail, sequential fixation with glutaraldehyde and freeze drying are comparable and often superior to commonly used techniques for preparing nematodes for SEM.     

Cryostat Microtomy of Lung Tissue in an Expanded State

A technique is reported for cryostat sectioning of lung tissue in an expanded state for use in viral immunofluorescence studies. A 1: 2 mixture of O.C.T. embedding compound and phosphate-buffered saline is injected intratracheally into fresh lung tissne. The lung tissue is frozen in liquid nitrogen and sectioned with a cryostat. Compared to other published reports of lung sectioning for immunofluorescence miscroscopy, this method has the advantages of bekg easy and quick, maintaining the lung sectiom in an expanded rather than collapsed state and avoiding contact with chemicals potentially capable of altering sensitive viral antigens.     

A method for orienting cryostat sections for three-dimensional reconstructions.

Tissue for cryostat sectioning is embedded together with reference tissue impaled on the needles of a specially constructed trident. To maintain spatial orientation, reference and experimental tissue are frozen in Tissue-Tek O. C. T. compound in the manner usual for enzyme histochemistry using a simply constructed carrier. The trident is removed by heating its handle, thus leaving holes in the reference tissue which in turn are used as reference points to orient successive sections with respect to each other.     

Autoradiography of Mobile, C14-Labeled Herbicides in Sections of Leaf Tissue

Fresh leaf tissue containing a soluble, C¹⁴-labeled herbicide was mounted in cold 1% gelatin on a holder, quick frozen in a cryostat, and cross sectioned at 16 μ with single-edge, stainless steel razor blades. The sections were transferred (without thawing) to cold (—10 C) microscope slides which had been partly covered with double-coated Scotch tape #665. The tissue was freeze-dried in a vacuum desiccator at—20 C then secured to the tape with pressure. Autoradiography was accomplished in a darkroom by covering the slides with dry, nuclear track emulsion films. These films were made by dipping 2 inch diameter wire loops into liquid emulsion, letting the film dry, and applying it by blowing it as it was placed against the tissue. After a 19 day exposure in light-tight boxes at 25-27 C the preparations were processed in the usual manner. The method-was used successfully to trace the movement of soluble, C¹⁴-labeled herbicides in leaf tissue without the loss of labeling material or artifacts caused by its diffusion. High resolution autoradiograms with low backgrounds were obtained.     

A Method For Orienting Cryostat Sections For Three-Dimensional Reconstructions

Tissue for cryostat sectioning is embedded together with reference tissue impaled on the needles of a specially constructed trident. To maintain spatial orientation, reference and experimental tissue are frozen in Tissue-Tek O. C. T. compound in the manner usual for enzyme histochemistry using a simply constructed carrier. The trident is removed by heating its handle, thus leaving holes in the reference tissue which in turn are used as reference points to orient successive sections with respect to each other.     

Autoradiography of Mobile,C14-Labeled Herbicides in Sections of Leaf Tissue

Fresh leaf tissue containing a soluble, C¹⁴-labeled herbicide was mounted in cold 1% gelatin on a holder, quick frozen in a cryostat, and cross sectioned at 16 μ with single-edge, stainless steel razor blades. The sections were transferred (without thawing) to cold (—10 C) microscope slides which had been partly covered with double-coated Scotch tape #665. The tissue was freeze-dried in a vacuum desiccator at—20 C then secured to the tape with pressure. Autoradiography was accomplished in a darkroom by covering the slides with dry, nuclear track emulsion films. These films were made by dipping 2 inch diameter wire loops into liquid emulsion, letting the film dry, and applying it by blowing it as it was placed against the tissue. After a 19 day exposure in light-tight boxes at 25-27 C the preparations were processed in the usual manner. The method-was used successfully to trace the movement of soluble, C¹⁴-labeled herbicides in leaf tissue without the loss of labeling material or artifacts caused by its diffusion. High resolution autoradiograms with low backgrounds were obtained.     

Adjustments in oxygen transport during head-out immersion in water at different temperatures

Respiratory gas exchange was investigated in human subjects immersed up to the shoulders in water at different temperatures (Tw = 25, 34, and 40 degrees C). Cardiac output (Qc) and pulmonary tissue volume (Vti) were measured by a rebreathing technique with the inert gas Freon 22, and O2 consumption (VO2) was determined by the closed-circuit technique. Arterial blood gases (PaO2, PaCO2) were analyzed by a micromethod, and alveolar gas (PAO2) was analyzed during quiet breathing with a mass spectrometer. The findings were as follows. 1) Immersion in a cold bath had no significant effect on Qc compared with the value measured at Tw = 34 degrees C, whereas immersion in a hot bath led to a considerable increase in Qc. Vti was not affected by immersion at any of the temperatures tested. 2) A large rise in metabolic rate VO2 was only observed at Tw = 25 degrees C (P less than 0.001). 3) Arterial blood gases were not significantly affected by immersion, whatever the water temperature. 4) O2 transport during immersion is affected by two main factors: hydrostatic pressure and temperature. Above neutral temperature, O2 transport is improved because of the marked increase in Qc resulting from the combined actions of hydrostatic counter pressure and body heating. Below neutral temperature, O2 transport is altered; an increase in O2 extraction of the tissue is even calculated.     

The influence of high pressure freezing on mammalian nerve tissue

Summary Vitrification of biological specimens in liquid nitrogen can be achieved under high pressure (2,100 bars). This procedure obviates the use of aldehyde fixation and cryoprotection (glycerol). The present work demonstrates its applicability to the freeze-etching of mammalian brain tissue. Freeze-fracture replicas from rat cerebellar cortex and subfornical organ prepared by this method are compared to conventionally processed material using aldehyde fixation, glycerination and freezing with Freon. The formation of large ice crystals is prevented in tissue blocks up to 0.5 mm thick; deep etching is markedly enhanced. Cytoplasmic microstructures such as mitochondrial cristae, microtubules and microfilaments, are readily observable against a finely granulated cytosol matrix. An additional advantage is the combined application with freeze-substitution.     

Hypothermia and hypoxia inhibit the Hering-Breüer reflex in the marsupial newborn

The effects of lowering body temperature (T(b)) on metabolic rate, ventilation, and the strength of the Hering-Breüer expiratory promoting reflex (HB reflex; determined from an inhibitory ratio calculated from volumetric measurements of the respiratory rhythm) were examined in 18-day-old ectothermic pouch young of the tammar wallaby during normoxia or hypoxia (10% O(2)). Hypoxia and hypothermia, either singularly or combined, depressed metabolic rate. At all T(b), the hypoxic hyperventilation was associated with a significant hyperpnea. At pouch T(b) (36.5 degrees C) during normoxia, inflation of the lungs with -5 or -10 cmH(2)O extrathoracic pressure induced a significant HB reflex. Exposure to cold reduced the strength of the reflex, almost abolishing it at 28 degrees C. For T(b) above 28 degrees C, the reflex in hypoxia was always less than the corresponding normoxic value. Taken in context with the changes in metabolic state that occurred, these data in the ectothermic marsupial newborn suggest that the decline in the HB reflex during moderate hypothermia is the result of a direct effect of T(b) on vagal mechanisms rather than a temperature-driven decline in metabolic rate that should have acted to strengthen the HB reflex. Therefore, it seems that inputs inhibitory to breathing are more negatively affected during cold than those inputs that are excitatory.     

Localization of Myoglobin in Striated Muscle of the Domestic Pig; Benzidine and NADH2-TR Reactions

Tissue blocks 1 cm³ from longissimus (white) and trapezius (red) muscles of adult pigs were fixed in phosphate-buffered 2.5% glutaraldehyde, pH 7.4, for 4 hr at about 25 C; washed 4 hr in running tap water, and immersed in 30% w/v sucrose solution for 16 hr or more. After freezing in liquid N₂, cryostat sections were cut and floated into saturated aqueous benzidine containing 0.15% H₂O₂ at 25 C for 30 min. Stained sections were washed in distilled water and mounted on slides with glycerol jelly. Three distinguishable gradiations of color intensity were found: strong, intermediate, and negative. The trapezius had a greater number of myoglobin-positive fibers than the longissimus muscle. Myoglobin-positive and myoglobin-negative staining occurred in red and white fibers, respectively; intermediates were apparently more closely related to the red than to the white fibers. The NADH₂TR reaction showed the same sites as did the benzidine reaction.     

The longest (A+T) and (G+C) blocks in the human and other genomes

We analysed complete or almost complete nucleotide sequences of the human, chimp, mouse, rat, chicken, dog, and other genomes to find that they contain extremely long (A+T) a (G+C) blocks that do not occur at all in the corresponding randomized sequences. The longest is an (A+T) block containing 1040 consecutive AT pairs that occurs in the 16th human chromosome. The longest human (G+C) block has 261 bp in length. About a half of the longest blocks occur in introns. The (A+T) blocks are discrete units whereas the (G+C) blocks are diffuse. They are imbedded in the genome through connectors longer than 1 kilobase where the (G+C) content gradually decreases to the value of 50%. Remarkably, the (A+T) as well as (G+C) blocks are substantially shorter in the chimp genome. Chicken is characteristic by very long (G+C) blocks that are even longer than in the human genome. Though much shorter, long (G+C) and especially (A+T) blocks occur in lower organisms as well, which means that AT and GC pair clustering is an ancient property that has evolved into large scales in higher eukaryote genomes and the human genome in particular. Very long (A+T) and (G+C) blocks confer specific biophysical properties on DNA that are likely to influence genome folding in cell nuclei and its functional properties.     

Elongation of synthetic RNA templates by hepatitis C virus NS5B polymerase

Here we examine the ability of seven, 3'-related, short synthetic RNAs to serve as templates for the hepatitis C virus (HCV) polymerase, non-structural protein 5B (NS5B). These RNAs, termed HL, range from 8 to 16 nucleotides in length, each with ACC at the 3' terminus. Interestingly HL12 and longer templates have a predicted secondary structure. Those with one or two unpaired adenylates at the 5'-end of a stem were increased in size by one or two nucleotides, respectively, following incubation with NS5B and UTP. Using labeled template RNA and cold UTP, extension in size could be inhibited by addition of non-labeled template of the same size. This template elongation was not inhibited by cold linear HL10 template unless pGpG was added. Fluorescence anisotropy demonstrated HL14, a template with secondary structure, bound with an apparent K(d) of 22 nm. A linear template, HL10, plus pGpG primer was bound by NS5B with a K(d) of 45 nm, whereas HL10 alone bound with an apparent K(d) of 182 nm. The amplitude of the template extension product was increased by a brief preincubation at 4 degrees C followed by incubation at 23 or 30 degrees C. The nucleotide-mediated increase in size occurred for both templates that required a mismatch or bulge at the 3'-end as well as for those without the mismatch. These results suggest an NS5B active site pocket can readily accommodate short templates with four or five base stems and initiate copy-back replication in the presence of a one nucleotide mismatch.     

Interactions of the cold shock protein CspB from Bacillus subtilis with single-stranded DNA. Importance of the T base content and position within the template

The cold shock protein CspB from Bacillus subtilis binds T-based single-stranded DNA (ssDNA) with high affinity (Lopez, M. M., Yutani, K., and Makhatadze, G. I. (1999) J. Biol. Chem. 274, 33601-33608). In this paper we report the results of CspB interactions with non-homogeneous ssDNA templates containing continuous and non-continuous stretches of T bases. The analysis of CspB-ssDNA interactions was performed using fluorescence spectroscopy, analytical centrifugation and isothermal titration calorimetry. We show that (i) there is a strong correlation between the CspB affinity and stoichiometry and the T content in the oligonucleotide that is independent of which other bases are incorporated into the sequence of ssDNA; (ii) the binding properties of CspB to ssDNA templates with continuous or non-continuous stretches of T bases with similar T content is very similar, and (iii) the mechanism of interaction between CspB and the T-based non-homogeneous ssDNA is mainly through the bases (a stretch of three T bases located in the middle of the ssDNA templates makes the binding independent of the ionic strength). The biological relevance of these results to the role of CspB as an RNA chaperone is discussed.     

An Ice-Solvent Method of Drying Frozen Tissue for Plant Cytology

A freeze-dry method where cold absolute ethanol is used as a dehydrating agent in place of vacuum dehydration has been applied to various plant materials with good cytological results. The method involves: (a) freezing rapidly small pieces of tissue 1 cubic mm or less in partly frozen isopentane cooled with liquid nitrogen, (b) transferring quickly to vials of cold absolute ethanol at -41° to -45°C, and (c) holding within this temperature range for 3 days to dissolve the ice. A simply constructed cryostat is used to maintain the vials of absolute alcohol and tissue at the cold temperature. This consists of a semi-frozen constant temperature bath of either 65% ethanol or pure diethyl oxalate in a tightly covered beaker which fits within a large dewar flask half filled with dry ice. The bath is arranged so that it will be on top of and in contact with the dry ice but properly insulated to prevent freezing completely.

The resulting dried tissue is very unstable in either water or hot absolute ethanol; therefore, to prevent loss of cytological detail during further processing, the tissue must be treated to render the proteins insoluble. Either (a) replace the cold absolute ethanol in the tissue vials with cold (approx. -40°C) 75% ethanol, warm slowly to 60°C, and hold for 1 hour, or (b) replace with cold acidulated 95% ethanol (100 ml. of 95% ethanol + 0.30 ml. of glacial acetic acid), warm to room temperature, and hold for 30 minutes. Following either treatment the tissues are dehydrated to absolute alcohol and embedded in paraffin by the usual technics. Sections are attached to slides by flattening over warm water and drying.

When epidermis from onion bulbs was used as a basis of comparison of fixed and living material with the phase-contrast microscope, the mitochondria, plastids, and other fine structures in fixed preparations appear to be nearly identical with the living. Fat droplets disappear. With larger tissues such as onion root tips, thin freehand sections must be prepared before freezing to obtain good cytological results. The application of the method to cytochemical studies is discussed and in many ways it seems to be as useful as the freeze vacuum-dry method.     

Cryostat Techniques: Methods for Improving Conservation and Sectioning of Tissue

Tissues have been conserved for satisfactory enzymatic histochemical assay for up to 12 mo by low temperature storage (in a dry ice chest or at -40° C) and by measures designed to offset the deleterious effects of sublimation of H₂O by using the following modifications of standard procedures (which include steps to give a mechanical support to otherwise fragmenting sections): 1. tissue blocks are coated with a polystyrene solution between each storage period; 2. modified bolts are used as tissue holders and types of bolt holders have been designed to fit on standard microtomes which permit manipulation of each tissue block independently of its mates on the same bolt holder, or the simultaneous cutting of all blocks on any one holder with each advance of the microtome feed; 3. tissues are coated with 20% polystyrene in methylene chloride prior to cutting and rubber cement painted on the slide as an adhesive. Lillie's 20% polystyrene diethylbenzene is used as a mounting medium. Other details of practical importance include the technique of freezing, control of moisture within a cryostat and on the microtome, and tests on histochemical procedures.