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PubMed2006

Association between seasonal changes in fatty-acid composition, expression of VLDL receptor and bovine sperm quality.

Reduced reproductive performance of dairy cows during the summer is often associated with elevated temperature. Semen collected and cryopreserved during the summer may be of low quality and might contribute to the compromised fertility of dairy cows during this season. The present study examined the association between seasonality, semen quality and its potential to survive cryopreservation. A comparison between semen collected during the summer (July to August) and that collected during the winter (November to December) revealed the summer semen to be inferior, as reflected by low motility and high mortality of sperm. Furthermore, samples that were defined as good quality had changes in lipid concentration and fatty-acid composition in both the seminal plasma and cell compartment. In particular, semen collected during the summer had reduced levels of polyunsaturated arachidonic acid (20:4; P<0.05) and decreased levels of linoleic acid (18:2; P<0.05) in the cell compartment; corresponding reductions in cholesterol (P<0.06) and fatty-acid concentrations (P<0.001) were detected in seminal plasma of semen collected during the summer. In addition, we provided the first evidence for the existence of a very-low-density lipoprotein receptor (VLDLr) in bovine sperm, suggesting a mechanism for sperm utilization of extracellular lipids. Interestingly, the expression of VLDLr was three-fold greater in samples collected during the winter than in those collected in the summer (P<0.01) and was negatively associated with saturated fatty-acid concentration (P<0.018) but not with that of cholesterol. An opposite pattern was noted for samples obtained during the summer; mRNA expression of VLDLr was negatively associated with cholesterol concentration (P<0.01) but not with that of saturated fatty acids. Such modifications associated with extracellular lipid utilization and fatty-acid composition might explain, in part, the reduced quality of summer semen.

Animals↗
PubMed2000

Predictors of success after embryo transfer: experience from a single provider.

OBJECTIVE: Our goal was to examine the variables present at the time of embryo transfer and to determine their effects on the clinical pregnancy rate. STUDY DESIGN: All fresh and frozen embryo transfers during a 3-year period in a university-based in vitro fertilization program were examined. Female age, previous in vitro fertilization attempt, diagnosis, embryo number and quality, transfer technique, and presence of a clinical pregnancy were recorded for each couple. Logistic regression analyses were performed both univariately and multivariately to determine the association between a clinical pregnancy and the independent variables. RESULTS: All transfers during the study period were included in the analysis. The four primary diagnoses were pelvic or tubal disease, male factor infertility, unexplained infertility, and endometriosis. The 46 frozen embryo transfers had a clinical pregnancy rate similar to that among the 159 fresh embryo transfers and were therefore included in the analysis. One variable was found to significantly affect the outcome, the number of high-grade embryos placed. The presence of a previous failed embryo transfer tended to lower the success rate for future attempts; however, this result did not reach statistical significance. The catheter type and the transfer difficulty did not affect outcome. CONCLUSION: The two most important variables for predicting a clinical pregnancy are a first-time transfer and the number of high-grade embryos placed. Neither the type of embryo transfer catheter used nor the diagnosis affected outcome. In this small sample difficult embryo transfers did not diminish the chance for a successful outcome.

Adult↗
PubMedDate not supplied

Standardization and quality control for the determination of alpha-glucosidase in seminal plasma.

We attempted to provide an instruction for the standardization of alpha-glucosidase level determination and quality controls within the same laboratory and among different laboratories. Each of 51 semen samples was divided into two aliquots, centrifuging at 1000 g for 10 min or 3000 g for 15 min. The alpha-glucosidase level and sperm concentration of each sample were assayed. The alpha-glucosidase level in seminal plasma obtained at 3000 g for 15 min centrifugation was significantly lower than that at 1000 g for 10 min (p = 0.001). An additional 6 samples of seminal plasma with or without phenylmethylsulfonyl fluoride (PMSF), obtained at 3000 g for 15 min centrifugation, were frozen for 20 days, and each of 6 samples was determined for their alpha-glucosidase levels after thawing every other day. There was no significant difference between alpha-glucosidase levels in seminal plasma regardless of the presence of PMSF. The alpha-glucosidase level increased with the length of abstinence period. In conclusion, centrifugal velocity and abstinence time should be standardized in the alpha-glucosidase determination. Frozen seminal plasma may serve as the sample for quality control among clinical laboratories.

Centrifugation↗
PubMed1998

Research notes: Fresh and frozen pools of chicken red blood cells as substrates for direct polymerase chain reaction.

A method is presented for reliable use of pooled chicken blood samples for estimation of microsatellite frequencies by direct polymerase chain reaction (PCR) amplification of DNA. This method overcomes the variability of hematocrit values in individual chickens and eliminates the step of DNA preparation. The estimated frequencies of polymorphic alleles in fresh and frozen pooled blood samples were similar to those obtained by calculating these frequencies from the individual genotyping. When frozen pooled blood samples are used, pools should be prepared prior to their freezing.

Animals↗
PubMed2006

Interference of blood cell lysis on routine coagulation testing.

CONTEXT: Preanalytical factors influencing the reliability of laboratory testing are commonplace. It is traditionally accepted that hemolytic samples are unsuitable for coagulation assays because of the release of hemoglobin, intracellular components, and thromboplastic substances from damaged blood cells. OBJECTIVE: To evaluate the influence of blood cell lysis on routine coagulation testing. DESIGN: Twelve aliquots prepared by serial dilutions of homologous lysated samples collected from 10 different subjects, and displaying a final percentage of lysis ranging from 0% to 9.1%, were tested for prothrombin time, activated partial thromboplastin time, fibrinogen, and dimerized plasmin fragment D. Lysis was achieved by subjecting citrated whole blood to a freeze-thaw cycle. OUTCOME MEASURES: Interference from blood cell lysis on routine coagulation testing. RESULTS: Statistically significant increases in prothrombin time and dimerized plasmin fragment D were observed in samples containing final lysate concentrations of 0.5% and 2.7% respectively, whereas significant decreases were observed in activated partial thromboplastin time and fibrinogen in samples containing a final lysate concentration of 0.9%. The current analytical quality specifications for desirable bias are +/-2.0% for prothrombin time, +/-2.3% for activated partial thromboplastin time, and +/-4.8% for fibrinogen. Percent variations from the baseline values exceeding the current analytical quality specifications for desirable bias were achieved for lysate concentrations of 0.9% (prothrombin time and activated partial thromboplastin time) and 1.8% (fibrinogen), corresponding to average free plasma hemoglobin concentrations of 1.7 and 3.4 g/L, respectively. CONCLUSION: Our results confirm that, although slightly hemolyzed specimens might still be analyzable, a moderate blood cell lysis, as low as 0.9%, influences the reliability of routine coagulation testing. Because the interference in coagulation assays has a wide interindividual bias, we do not recommend lysis correction and we suggest that the most appropriate corrective measure should be free hemoglobin quantification and sample recollection.

Artifacts↗
PubMedDate not supplied

Intact PTH study: shipping frozen vs. unfrozen samples.

DCI Laboratory, Nashville TN compared INCSTAR N-tact PTH SP Kit (INCSTAR Corp., Stillwater, MN, USA) results from renal dialysis patients analyzed on frozen versus unfrozen samples; unfrozen samples were analyzed at 24, 48, and 72 hours post-collection. There was no statistical or clinical difference in the results of the frozen samples and unfrozen samples analyzed at 24 hours. Clinics may now ship unfrozen samples if they arrive within 24 hours.

Biomarkers↗
PubMed2006

Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus).

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25 degrees C or after slow cooling to 5 degrees C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (+/- s.e.m.) 43.6 +/- 14.2 x 10(6) motile spermatozoa with 33.5 +/- 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5 degrees C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25 degrees C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

Acrosome↗
PubMed2002

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers.

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.

Cell Adhesion↗
PubMed2004

Influence of cryopreserved olfactory ensheathing cells transplantation on axonal regeneration in spinal cord of adult rats.

OBJECTIVE: To observe the effects of cryopreserved olfactory ensheathing cells (OECs) transplantation on axonal regeneration and functional recovery following spinal cord injury in adult rats. METHODS: Twenty-four rats were divided into experimental and control groups, each group having 12 rats. The spinal cord injury was established by transecting the spinal cord at T10 level with microsurgery scissors. OECs were purified from SD rat olfactory bulb and cultured in DMEM (Dulbecco's minimum essential medium) and cryopreserved (-120 degree) for two weeks. OECs suspension [(1-1.4)x10(5)/ul] was transplanted into transected spinal cord, while the DMEM solution was injected instead in the control group. At 6 and 12 weeks after transplantation, the rats were evaluated with climbing test and MEP (moter evoked potentials) monitoring. The samples of spinal cord were procured and studied with histological and immunohistochemical stainings. RESULTS: At 6 weeks after transplantation, all of the rats in both transplanted and control groups were paraplegic, and MEPs could not be recorded. Morphology of transplanted OECs was normal, and OECs were interfused with host well. Axons could regrow into gap tissue between the spinal cords. Both OECs and regrown axons were immunoreactive for MBP. No regrown axons were found in the control group. At 12 weeks after transplantation, 2 rats (2/7) had lower extremities muscle contraction, 2 rats (2/7) had hip and/or knee active movement, and MEP of 5 rats (5/7) could be recorded in the calf in the transplantation group. None of the rats (7/7) in the control group had functional improvement, and none had MEPs recorded. In the transplanted group, histological and immunohistochemical methods showed the number of transplanted OECs reduced and some regrown axons had reached the end of transected spinal cord. However, no regrown axons could be seen except scar formation in the control group. CONCLUSIONS: Cryopreserved OECs could integrated with the host and promote regrowing axons across the transected spinal cord ends.

Animals↗
PubMedDate not supplied

Effects of thawing procedure on postthawed in vitro viability and in vivo fertility of red deer epididymal spermatozoa cryopreserved at -196 degrees C.

In this study, we have determined the effects of individual factor and thawing procedure on in vitro viability and in vivo fertility of frozen-thawed red deer epididymal spermatozoa. The spermatozoa that were collected from 13 Iberian deer stags were diluted at room temperature in a Triladyl-20% egg yolk medium and frozen in nitrogen vapors. In the first experimental series, sperm samples were collected from 10 mature stags. For thawing, the frozen straws were subjected to 3 different procedures: I (37 degrees C for 20 seconds), II (60 degrees C for 8 seconds) and III (70 degrees C for 5 seconds). Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SM) and of plasma membrane and acrosome (NAR) integrities. Statistically significant variations were found (P <.05) between stags for most of the seminal parameters evaluated. The thawing procedure did not have an effect on the seminal characteristics evaluated after this process, except for SM (P <.05), with the best overall recovery rates after freezing and thawing found with the use of protocol I. Our results also show a differential resistance to return to isosmotic conditions of spermatozoa thawed using the different thawing protocols. In the second experimental series (insemination artificial trial), with spermatozoa from 3 stags, results of fertility were statistically higher (69.7% vs 42.4%, P =.014) when spermatozoa were thawed at 37 degrees C for 20 seconds than were warmed at 60 degrees C for 8 seconds. Therefore, thawing protocol I, which provides slow thawing rates, was the most beneficial for epididymal spermatozoa thawing of the cervid subspecies analyzed in this study. In summary, high in vitro survival and in vivo fertility of frozen-thawed deer epididymal spermatozoa were dependent on warming rates, but each stag exhibited its own sensitivity to cryopreservation.

Animals↗
PubMed2005

Vitrification of calf oocytes: effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development.

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.

Actins↗
PubMed2007

Morphological changes in mouse embryos cryopreserved by different techniques.

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing.

Animals↗
PubMed1997

Cryopreservation of human spermatozoa: comparison of two cryoprotectants and two freezing methods.

A prospective experimental study was performed to compare sperm cryosurvival rate between two kinds of cyroprotective media: citrate-egg yolk-glycerol (CEG) and TEST-citrate-egg yolk-glycerol (TEST-EYG), and between two freezing methods: noncontrolled rate (NCR) freezing and computer controlled rate (CR) freezing. One hundred semen samples were included in the study. Each sample was divided into two parts, which were prepared as two CEG and two TEST-EYG mixture straws, then one straw from each mixture was frozen by the NCR-freezing method and the complementary straw was frozen by the CR-freezing method. After one week of cryostorage, the straws were thawed and the post-thaw sperm survival was assessed. As the results, the post-thaw sperm survival rate of the CEG media group was slightly higher than the TEST-EYG group in both the NCR-freezing method (51.2% vs 47.0%, p < 0.05) and the CR-freezing method (48.2% vs 43.4%, p < 0.05); and similarly; the NCR-freezing group yielded a slightly higher post-thaw sperm survival rate than the CR-freezing group in both the CEG media used (51.2% vs 48.2%, p < 0.05) and the TEST-EYG media used (47.0% vs 43.4%, p < 0.05). However, these did not have clinical significance because the differences were too small. In conclusion, the use of either CEG or TEST-EYG media, and either the NCR or CR-freezing method did not have clinical differences on the post-thaw sperm survival.

Adult↗
PubMed2004

Sperm cryopreservation of a live-bearing fish, the platyfish Xiphophorus couchianus.

The objectives of this study were to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio, as well as somatic relationships of body length, body weight, and testis weight to sperm density in the platyfish Xiphophorus couchianus. Sperm motility and survival duration after thawing were significantly different between cryopreservation with dimethyl sulfoxide (DMSO) and glycerol, with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) across a range of 240-300 mOsm/kg. Samples cooled from 5 to -80 degrees C at 25 degrees C/min yielded the highest post-thaw motility, although no significant difference was found for cooling rates across the range of 20-30 degrees C/min. In addition, the highest motility after thawing was found in samples equilibrated from 10 to 30 min with 14% glycerol and cooled at 25 degrees C/min. The post-thaw motility declined rapidly with use of 10% glycerol and cooling at 5 degrees C/min across the equilibration range of 10 min to 2h. Sperm motility with a dilution ratio of sperm to extender of 1:10 was not different at 10 min after thawing with those samples at greater dilutions, but declined significantly from Day 1 after thawing and showed lower survival duration when stored at 4 degrees C. However, the additional dilution of sperm solutions with HBSS (300 mOsm/kg) immediately after thawing significantly slowed the decline of motility and prolonged the duration of survival. Based on the above findings, the highest average sperm motility (78+/-3 %) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsm/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 25 degrees C/min from 5 to -80 degrees C before plunging into liquid nitrogen, and thawed at 40 degrees C in a water bath for 7 s. If diluted within 5 h after thawing, sperm frozen by the above protocol retained continuous motility for 15 days when stored at 4 degrees C.

Animals↗
PubMed2005

A new and simple method to evaluate early membrane changes in frozen-thawed boar spermatozoa.

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols and has previously been studied in the pig species using annexin-V detection of phosphatidylserine translocation. In the present study we designed a new assay to detect these changes in boar spermatozoa, based on the slight increase of sperm membrane permeability occurring during the early stages of cryoinjury, using the combination of three fluorescent probes, SNARF-1, YO-PRO-1 and ethidium homodimer. Four ejaculates from five different boars were frozen-thawed and flow cytometrically (FC) evaluated as paired samples. One of the samples was assayed using the annexin-V/propidium iodide staining and the other sample was evaluated using the new triple staining. Using this combination of probes, four sperm subpopulations were easily detected: viable, with stable membranes (SNARF-1 positive cells), and three with compromised membranes, one of YO-PRO-1+/Eth- cells, one ethidium homodimer+ spermatozoa and, finally spermatozoa stained both with YO-PRO-1 and ethidium homodimer (YO-PRO-1+/Eth+). The latter three categories corresponded to dead spermatozoa, but with different degree of membrane damage, being YO-PRO+/Eth- an earlier stage of membrane destabilization, (manifested by an increase in membrane permeability, while still maintaining membrane integrity) than YO-PRO+/Eth+. A method agreement analysis between both methods was performed revealing good agreement, although the percentage of live cells was 9.44% larger for the triple stain than the annexin-V assay. The new assay stained all sperm sub-populations present in the sample, making it especially suitable for both fluorescence microscopy and flow cytometry, facilitating the exclusion of debris and egg-yolk particles when using FC.

Animals↗
PubMed1997

Umbilical cord blood collection and separation for haematopoietic progenitor cell banking.

Cord blood transplantation has been proven to be a suitable form of treatment for a variety of diseases in childhood and more recently in an increasing number of adult patients. Banks of cord blood cryopreserved after HLA testing are required in order to provide various HLA types for unrelated transplantation. To optimize storage space cord blood needs to be stored as a separated product. Several early methods of cord blood separation resulted in a significant loss of progenitor cells. We used a separation procedure where the donation was separated by centrifugation into a buffy coat fraction, a red cell fraction, and a plasma fraction. Twenty-five samples, (mean initial volume 81 ml) were assessed. Nucleated cells were recovered in the buffy coat fraction. Recoveries of nucleated cell count, total progenitors and CD34-positive cells in the buffy coat were 90%, 88% and 100%, respectively. The buffy fraction was tested for sterility by aerobic and anaerobic culture. Using this closed bag system, volume reduction was achieved while maintaining sterility and retaining progenitor cells in a final mean buffy coat volume of 44 ml. Red cell and plasma fractions were available for ABO grouping, virology testing and cryopreservation. The results show that cord blood can be effectively volume-reduced using simple and readily available blood banking techniques.

Adult↗
PubMedDate not supplied

An improved cryopreservation protocol for pineapple apices using encapsulation-vitrification.

Several modifications to the cryogenic protocols previously described for pineapple apices were performed using vitrification and encapsulation-vitrification. Pregrowth of apices in sucrose-proline before loading significantly reduced the exposure duration to PVS2 and PVS3 required for successful cryopreservation. Encapsulation and treatments with PVS3 at 0 degree C gave the highest survival before and after cooling. Optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3 percent) followed by a 2-d preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h and then transfer to 0.3 M sucrose + 0.3 M proline for an additional 24 h. After preculture, samples were loaded in 0.75 M sucrose + 1 M glycerol solution at room temperature (25 min) and dehydrated with PVS3 at 0 degree C for 60 min before immersion into liquid nitrogen. Following this procedure 54 percent and 83 percent of apices from MD-2 and Puerto Rico varieties respectively survived.

Ananas↗
PubMed1995

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Aged↗