Histochemical Demonstration of Carbonic Anhydrase Activity

Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10-30 minutes were incubated for 30-45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl₂·4H₂O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15-20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.     

A modification in technology improves the specificity of the Du Pont de Nemours immunoenzyme kit for the detection of anti-HIV antibodies

Du Pont De Nemours perfected a new enzyme immunoassay for the screening of anti-HIV 1 antibodies, characterized by its rapidity: 2 hours of incubation, when the normal procedure needed 3 hours 30 minutes of incubation. We tested with the normal procedure and the shortened one 4,025 randomly selected blood donors. 55 samples of the SNTS panel and 10 sera representing the Western-Blot reference panel of the "Retrovirus" study group of the SNTS. We can say that shortening the incubation time results in certain advantages opposite the previous procedure: time saving (1 hour 30 minutes); improved specificity: the rate of false positive, 0.37% with the normal method, drops to 0.07% with the short one; good sensitivity, since all the individual positive samples were detected but it seems slightly less sensitive than the normal method for the diluted positive samples.     

Lowicryl K4M embedding of brain tissue for immunogold electron microscopy

We present methods for embedding brain tissue in Lowicryl K4M embedding medium and localizing antigens using postembedding immunogold techniques. After perfusion fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, blocks of rat brain were placed in 2% aqueous uranyl acetate for 1 hour, dehydrated in 50%, 70%, and 95% ethanol, infiltrated with Lowicryl/ethanol mixtures (1:2 for 10 min, 1:1 for 15 min) and 100% Lowicryl (20 min and 25 min). Polymerization was carried out under UV light for 24-48 hours at room temperature. Several neural antigens, including three different synaptic vesicle proteins and an enzyme associated with the postsynaptic density, were localized by this technique, indicating that this procedure may have wide applicability.     

Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed tissue

Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO3, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

Toluidine blue binding capacity of heterochromatin and euchromatin of Triatoma injestans Klug

Summary Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO₃, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

S／D处理血浆过程中的脂包膜病毒灭活试验观察

通过有机溶剂/去污剂对血浆中指示病毒VSV灭活的观察评估有机溶剂/去污剂对脂包膜病毒死活的效果。血浆样品与VSV病毒按9:1混合,然后用1％TNBP/1％ Triton X-100在30℃处理4h,测定开始样品中的病毒总量和S/D处理后不同时间取样内的病毒总量。实验中样品内加入1％TNBP/1％ Triton X-10015min后VSV病毒已全部灭活,灭活效果≥6.0log。按所述S/D处理方法可以完全灭活血浆内所有的脂包膜病毒而没有主要血浆蛋白的损失。     

A simple method for the preparation of paraffin-embedded cell blocks from fine needle aspirates, effusions and brushings

A simple method for the preparation of paraffin-embedded cell blocks from cytologic specimens obtained by fine needle aspiration, by brushing or from effusions is described. The cells are fixed in suspension in 50% ethanol for one hour and pelleted by centrifugation in a 50-mL plastic tube. The fixative is removed, and the pellet is suspended in 3 mL of acetone for dehydration for ten minutes and thereafter repelleted. The acetone is then removed, and the cell pellet is dried at 60 degrees C for one hour. Melted paraffin is added onto the dry warmed cell mass and allowed to solidify at room temperature. A conical paraffin block with the cells in the top is obtained and can be handled as a routine tissue block.     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

Permanent Mounts Of Chromosomes After 8-Oxyquinoline and Squashing

Fresh young root tips or free-hand cross sections thereof were placed in 0.002 M 8-oxyquinoline (aq.) at 10-14^oC. for 3 hours. After rinsing in water 1-2 minutes, they were soaked in N HC1 at 55^oC. for 25 minutes, rinsed again and squashed under a cover glass on a dry slide. Slide and cover glass were separated by placing in 70% alcohol and allowed to remain therein at least 0.5 hour after separation. Both slide and cover glass were passed through 50% and 30% alcohol to water and stained by the Feulgen procedure (without further hydrolysis) or with crystal violet after mordanting in 1% chromic acid overnight and washing in running water 3-4 hours. Dehydration and mounting in balsam completed the process. The smear on the slide was covered with a clean cover glass and the cover glass, bearing stained material, mounted separately.     

The Dehydration of Methylene Blue Stained Material Without Loss of Dye

A method is given for dehydrating methylene blue stained protozoan smears which should be applicable to the dehydration of tissues stained intra vitam with methylene blue. The procedure is: Wash with distilled water, place in tertiary butyl alcohol for 1 to 2 minutes, then in three or more changes of tertiary butyl alcohol for 15 minutes to an hour each, and mount directly in balsam or pass thru two changes of xylene before mounting.     

Optimal prefixation of cells to demonstrate apoptosis by the TUNEL method.

OBJECTIVE: To determine the optimal fixation method for cultured human ovarian cancer cell line SHIN-3 to document cisplatin-induced apoptosis by the terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling (TUNEL) assay. STUDY DESIGN: Cisplatin-treated cancer cell suspensions were (1) fixed in 4% buffered formalin for 10 minutes (BF group); (2) treated with 2% Carbowax in 50% ethanol (CW) for 10 minutes and then fixed in 100% ethanol for one hour (CW + ET); or (3) treated with CW for 10 minutes and then fixed in 4% buffered formalin for one hour (CW + BF). Cell morphology, adhesion to the glass slides and TUNEL reactivity were compared among the three groups. The effects of prolonged prefixation of cell suspensions in CW and of the postfixation of cell smears in BF for one, three and seven days were also examined. RESULTS: CW + BF treatment yielded satisfactory cell morphology, minimum cell loss and an excellent TUNEL reaction. However, prolonged prefixation in CW resulted in cell shrinkage. CONCLUSION: CW + BF treatment can be widely recommended for use with cytologic preparations for the TUNEL assay.     

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.     

A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

A New Method Using Hexamethyldisilazane for Preparation of Soft Insect Tissues for Scanning Electron Microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

An improved dispersed adrenal cell assay for corticotropin in rat plasma

The present study was designed to improve the dispersed adrenal cell technique for determining adrenocorticotrophic hormone (ACTH) concentrations in small amounts of rat plasma. Priming with ACTH, incubation with methyl-isobutylxanthine, or dexamethasone pre-treatment were employed as modifications. Of these, only dexamethasone pre-treatment increased the sensitivity of the assay. The adrenal fragments obtained from 10-12 adult male rats pre-treated with dexamethasone (100 micrograms/kg B.W.) one hour before sacrifice, were digested with collagenase and deoxyribonuclease solution for 30 minutes. The dispersed cells were collected by centrifugation and resuspended in Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.5% bovine serum albumin. Aliquots of cell suspension (3-4 X 10(4)/tube) were incubated with various doses of ACTH1-24 or the eluate of plasma samples at 37 degrees C for 2 hours in an atmosphere of 95% O2/5% CO2 in a Dubnoff shaker. The quantity of corticosterone produced was measured fluorimetrically. The assay is precise (lambda = 0.06), extremely sensitive (10 fg/tube), and convenient. One skilled technician can handle 15 to 20 plasma samples per day using 10 rats as the source of assay cells. ACTH can be measured in as little as 10-50 microliters of eluate.     

Silver Staining of Nerve Fibers in Cardiac Tissue

Fresh hearts of dog were perfused through the coronary vessels with 1000 ml. of fixative (chloral hydrate, 5 g. per 100 ml. of 70% ethyl alcohol) and blocks of tissue 2 × 5 mm. from epicardium to endocardium fixed 48 hours in the same fixative. The blocks were placed in 95% alcohol containing 0.3% addition of strong ammonia for 4 hours, followed by 2 changes of plain 95% alcohol of 1 hour each, then cleared and infiltrated with paraffin. Mounted sections 12-15 µ thick were incubated in 1% silver proteinate (obtained from Serumvertrieb, Marburg, Germany)² at 38° C. for 48 hours in the presence of 10 g. of 15 gauge copper wire per 200 ml. of solution. The slides were rinsed gently in 3 changes of distilled water for 2 minutes, 1 minute and 1 minute, respectively, and reduced in 1% hydroquinone and 5% sodium sulfite for 5 minutes. They were washed 5 minutes in tap water and 5 minutes in 2 changes of distilled water and toned 3-5 minutes in 0.25% gold chloride, rinsed in distilled water 10 seconds, reduced 10 seconds in 1 % oxalic acid, rinsed 1 minute, fixed in 5% sodium thiosulfate 5 minutes, washed in tap water through 3 changes, dehydrated, cleared and covered. All solutions were made with distilled water except where otherwise specified. The results gave good impregnation of fine nerve fibers without the usual confusing staining of reticular tissue.     

A Differential Stain for Rubber in Guayule

The following schedule, which combines an intense blue stain for rubber with sharply contrasting red counterstains, has been found satisfactory for use in an anatomical study of rubber deposition in guayule: Cut fresh or fixed sections about 50 to 100 % thick, transfer to 50% ethanol. Extract with acetone 5 minutes, treat with 1% NaOCl 5 minutes, saponify with 10% KOH in 95% ethanol 15 minutes, rinse 3 times with 50% ethanol, stain in oil blue NA (Calco) with safranin and Congo red 30 minutes at 55° C. Rinse in 50% ethanol 2 (or more) times to remove excess stain and mount in Karo syrup.     

Duration of Last Attack in 998 Fatal Cases of Coronary Artery Disease and its Relation to Possible Cardiac Resuscitation

All the deaths attributed to coronary artery disease and occurring in Belfast during one year were studied.The frequency distributions of the cases by interval of time between onset of the last attack and death are given for those not admitted to hospital, for those admitted to hospital, and for those already in hospital for some other cause of illness.Sixty per cent. of all the deaths occurred outside hospital. This indicates that the problem of cardiac resuscitation in coronary artery disease is to a considerable extent an extra-hospital one.Twenty-seven per cent. of the men and 22% of the women died within 15 minutes, but the median period of survival was 3 hours 30 minutes for men and 6 hours 18 minutes for women.The median time interval from the onset of the attack to sending for medical aid was 1 hour 17 minutes for men and 1 hour 6 minutes for women, and from summoning medical aid to sending for the ambulance 59 minutes for men and 1 hour 26 minutes for women. Ninety-six per cent. of the ambulance journeys to the patient were accomplished in less than 20 minutes.It was found among men, but not among women, that the duration of survival tended to be longer in older patients and in second or subsequent attacks.Of the 596 who did not gain admission to hospital 229 (23% of all the 998 patients) were known to have survived for more than half an hour after the onset of the fatal attack; 182 (18%) survived for more than one hour; and 143 (14%) survived for more than two hours. It is among these that there would appear to be special scope for the cardiac ambulance, providing that medical aid is sought and the ambulance is summoned without delay.     

Acute ethanol administration and transient ischemia: a behavioral and neuropathological study

A pressing clinical question is how acute ethanol exposure might alter the outcome of a simultaneous transient ischemic attack (TIA), since ethanol is known to dysregulate key intermediary metabolites post-ischemia. Mongolian gerbils were administered ethanol (1 or 4 g/kg, s.c.) 1 hour before induction of transient ischemia, via bilateral carotid occlusions of 5 minutes duration. A control group was administered isotonic saline and rendered ischemic. All animals were maintained normothermic during the ischemic procedure. Subjects underwent behavioral assay of acquisition to the water maze 7 days after recovery from the surgery, and neuropathological examination 1-month after the ischemic brain insult. There were no behavioral or neuropathological between-group differences suggesting that mechanisms other than adverse ethanol-induced perturbations of ischemic processes predominate in mediating epidemiological findings of elevated stroke morbidity with high ethanol consumption.     

A rapid method for demonstrating skeletal muscle motor innervation in frozen sections.

Longitudinal 50-100 mum-thick frozen sections of muscle are picked up on slides coated with 3% EDTA and after drying are incubated to demonstrate acetylcholinesterase. Subsequent incubation in 0.5% K3Fe(CN)6 is followed by fixation for 30 minutes in formol-calcium or formol-saline. After washing, the slides are incubated in 20% aqueous AgNO3 containing 0.1% CuSO4 for 2-30 minutes at 37 C. Following development in a 1% solution of quinol (w/v) 5% with respect to NaSO3 (w/v), axons and subneural apparatus stain dark brown to black in contrast to the less well stained muscle fibers and nuclei. This procedure permits study of the pattern of neuromuscular innervation in skeletal muscle 3 1/2-4 hours after receipt of a sample, and makes possible determination of the terminal innervation ratio.