Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

Acid Ethanol Fixation and Polyester Wax Embedding Combines Preservation of Antigenic Determinants with Good Morphology and Enables Simultaneous Bromodeoxyuridine (BRDU) Labeling

Lymphoid tissue, and/or isolated peripheral mononuclear blood cells were fixed in acid ethanol and embedded in polyester wax (melting point 37 C). The excellent cytomorphol-ogy obtained allowed distinguishing different types of individual lymphoid and nonlymphoid cells. Furthermore, this procedure was satisfactory in the immunophenotyping of histiocytes, endothelial, mesenchymal, epithelial cells, different (sub-) types of lymphocytes and also of lym-phokine activated killer (LAK) cells. The staining patterns obtained with the different poly- and monoclonal antibodies on polyester wax sections were not only analogous to those obtained on frozen sections, but cells which had incorporated bromodeoxyuridine could be double labeled with specific antiserum.     

Carbowax Embedding for Obtaining Thin Tissue Sections and Study of Intracellular Lipids

Carbowax, a water soluble wax, as an embedding agent is a valuable adjunct to the armamentarium of the tissue technologist. This report is intended to supplement previous publications on the use of Carbowax and to indicate die necessity for preheating and variation of Carbowax mixtures according to the climate.

Carbowax embedding provides an easy means for obtaining tissue sections 1 to 3 μ in thickness either with or without previous exposure to fat solvents. These sections are admirably suited for cytological study, particularly of intracellular lipoids.     

Alkaline and Acid Phosphatase Demonstration in Human Bone and Cartilage: Effects of Fixation Interval and Methacrylate Embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

An Improved Method for the Fixation, Embedding and Immunofluorescence Labeling of Resin-Embedded Plant Tissue

The use of antibodies as direct probes for specific macromolecules in plant cells and tissue is a well-established and extremely powerful technique and is of particular use in the post-genomics era. In this paper, we present an improved fixation, embedding, and immunofluorescence technique suitable for fixing “difficult” plant tissues such as pistils and inflorescence stems, which possess many trichomes and a thick hydrophobic cuticle. The key modification of the fixative used in the current study was the addition of a small amount of sucrose, CaCl₂, and detergent into a 4% (v/v) formaldehyde and 1% (v/v) glutaraldehyde mixture without the requirement to vacuum infiltrate. The modified immunofluorescence labeling method featured an amended blocking buffer, increased number of washing steps, and the use of an aqueous mounting medium which produced intense immunolabeling signals with extremely low background. Moreover, the immunocytochemistry methodology described in this study has proven to be suitable for use on two widely studied plant species, namely, Vicia faba and Arabidopsis thaliana, and may, therefore, be applicable for use in studies of a wide range of angiosperms.     

Effect on Tissue Volume of Various Methods of Fixation, Dehydration, and Embedding

A new indirect method is described for following volume changes of homogeneous pieces of tissue during fixation, dehydration and embedding, and area changes during sectioning, staining and mounting. Pieces of rabbit kidney cortex were compared after fixation in Destin's, Orth's, Petrunkevitch's cupric-paranitrophenol, Bouin's, SUSA, Zenker-formol, 10% formalin in distilled water, formalin in saline, Burke's pyridine formalin, CaCOy neutralized formalin, MgCO₃-neutralized formalin, Bensley's vacuum distilled neutral formalin in distilled water, and Bensley's neutral formalin in saline; during dehydration in ethyl alcohol, dioxan, and tertiary butyl alcohol and clearing in xylol and chloroform; and after infiltration and embedding with parowax, with paraffin-nitrocellulose double embedding technic, with hot nitrocellulose, and with cold nitrocellulose. The H-ion concentration of these fixatives was followed during tissue fixation.

Altho all fixatives showed specific merences, SUSA and Bouin's gave the best general results and neutral formalin mixtures, especially pyridine-formalin, the poorest. Isotonic saline was found superior to distilled water as a formalin diluent, reducing tissue swelling during formalin fixation. Reagents producing marked decreases in tissue volume render such tissues less susceptible to shrinkage during subsequent procedures. Shrinkage of tissue during dehydration and infiltration with hot parffin may exceed that produced by fixation alone. Excessive heat causes tissue distortion and shrinkage. Inijltration with hot paran causes considerable shrinkage, with hot nitrocellulose Iess, and with cold nitrocellulose the least sbrinkage.     

An Improved Method for Rapid Fixation and Embedding of Pteridophyte Plant Materials

A rapid method for fixation and embedding of plant materials, especially pterido-phytes, is suggested. Addition of tannic acid following osmication improved the visualization of membranes. Staining en bloc with uranyl acetate between osmication and tannic acid is suggested for tissues infected with fungi and bacteria.     

非特异性酯酶及酸性和碱性磷酸酶在奥尼罗非鱼消化道的分布

本研究利用石蜡切片结合组织化学方法研究奥尼罗非鱼(Oreochromis niloticus♀×O. aureus♂)消化道内非特异性酯酶、酸性磷酸酶和碱性磷酸酶的分布特点。结果发现,非特异性酯酶大量密集分布在前肠,其次为胃、食管和中肠,极少数零散分布在后肠。酸性磷酸酶主要分布于奥尼罗非鱼前肠,其次为中肠和胃,极少数分布在食管和后肠。碱性磷酸酶大量密集分布在奥尼罗非鱼前肠和中肠,其次为胃、食管和后肠。结果表明,奥尼罗非鱼前肠、中肠、食管和胃有较强的吸收和消化脂质功能,前肠、中肠和胃是蛋白质吸收和消化的主要部位,前肠和中肠是营养物质吸收的主要部位。     

The Hazard of Acid Differentiation in Gomori's Method for Acid Phosphatase

In his original method for the histochemical demonstration of acid phosphatase, Gomori prescribed differentiation of incubated sections by rinsing them in 14% aqueous acetic acid, to remove the nonspecifically precipitated lead deposits. According to him, the enzymatically produced lead phosphate is not washed out by this procedure. As a result of recent improvements in tissue preparation and shorter incubation time, this staining reaction as it is used now is quite sensitive to an acetic acid wash. If this wash is used as recommended originally, it may completely abolish a truly positive reaction. To avoid falsely negative results, and to compare sections of normal and pathological tissue, omission of this differentiation by acetic acid is essential. The risk of mistaking nonspecific lead precipitates in the interpretation of a positive reaction is very small, and can be avoided by running a negative control slide in which no lead phosphate can be produced enzymatically.     

铅对鲫鱼碱性磷酸酶和酸性磷酸酶活性的影响

在实验条件下,将鲫鱼置于1、10、40 mg/L的Pb2 溶液中静态染毒,分别在24 h、48 h、96 h、144 h测定鳃、肝、胰、肾、脑组织的碱性磷酸酶(AKP)和酸性磷酸酶(ACP)活性.结果 表明,鲫鱼鳃、肝、胰、肾、脑组织中碱性磷酸酶和酸性磷酸酶的活性随着铅浓度的升高和染毒时间的延长均呈下降趋势,其中以肾脏和脑下降最明显.     

高温酸性磷酸酶acp基因的克隆、表达和功能的初步鉴定

从高温塘泥(温度在40～85℃之间)中分离到一组嗜热微生物,提取环境基因组DNA,通过随机测序,发现许多基因与Thermus属的DNA相似性很高,根据Genbank上Thermus thermophilusHB8基因组序列中推定的酸性磷酸酶基因(TTHA1616),设计引物,PCR扩增得到一段798bp的DNA序列,测序结果与Thermus thermophilusHB8基因组中酸性磷酸酶基因相似性达98%,含有完整的酸性磷酸酶保守区,于是将该基因在大肠杆菌中表达,得到一条约33KD的特异蛋白条带,酶活检测证实该推定蛋白具有酸性磷酸酶活力。     

Human Prostatic Acid Phosphatase,an Authentic Tyrosine Phosphatase,Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr^1221/2 correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr^1221/2. Concurrently, Tyr³¹⁷ phosphorylation of p52^Shc, proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr^1221/2. Its downstream p52^Shc, ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr^1221/2 phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr^1221/2 and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.     

Punched-Out Agar Petri Plates for Systematic Fixation and Embedding of Multiple Small Tissue Samples

A 5% agar solution is poured into Petri dishes, jelled, and a pattern of cylindrical holes punched out according to the number and size of tissue samples to be processed. These punched-out Petri plates withstand neutral formalin, water, benzene, and paraffin immersion at 60 C; thus providing an adaptable container for processing small tissue samples. Reduced handling of the tissues, minimized risk of loss, and easy identification of samples by means of the map-like arrangement of the holes are among the virtues of such plates. An optimal fixative-volume: tissue-weight ratio can be attained by using wells of varying diameter and depth according to the sample size.     

A Filter Method for Processing Small Samples of Marine Ova Through Fixation, Dehydration, and Embedding Without Changing Containers

Free-floating cells can be fixed, dehydrated, and embedded in a single container. The container was constructed from stainless steel, and the paraffin block formed by the shape and size of a container was perfect for microtoming. Eight containers were embedded in a fiberglass holder. This holder was designed so that it could be used with a 47 mm Millipore filter. Cells were pipetted into the top of a container while the Millipore filter sealed the bottom; thus the cells were retained on the filter while fluids were allowed to pass through it. The exposure of the cells to histological reagents was regulated by applying a vacuum to control the rate of flow through the filter.     

Effects of Gibberellic Acid on the Activation, Synthesis, and Release of Acid Phosphatase in Barley Seed

Acid phosphatase activity was present in unimbibed barley seed,but rose during incubation of embryoless half-seeds and isolatedaleurone layers, and was further increased by 10^–6 M gibberellicacid (GA₃). Release of total acid phosphatase activity fromhalf-seeds and aleurone layers was markedly enhanced by GA₃.Inhibitor studies with cycloheximide and actinomycin D suggestedthat de novo synthesis of acid phosphatase occurred followingimbibition. Gel nitration, electrophoresis, and [¹⁴C]leucineincorporation studies revealed that a single molecular formof acid phosphatase was present in dry seed, whereas on incubationtwo further forms arose. A proportion of the three molecularforms of the enzyme was synthesized de novo. Gibberellic acidstimulated activation, but not de novo synthesis, of all threemolecular forms of acid phosphatase. Although a small amountof one of the molecular forms was secreted in the absence ofGA₃, the presence of gibberellin greatly increased secretionof the same form of acid phosphatase.     

Purification and Characterization of a Potato Tuber Acid Phosphatase Having Significant Phosphotyrosine Phosphatase Activity

The major acid phosphatase (APase) from potato (Solanum tuberosom L. cv Chiefton) tubers has been purified 2289-fold to near homogeneity and a final O-phospho-L-tyrosine (P-Tyr) hydrolyzing specific activity of 1917 [mu]mol Pi produced min-1 mg-1 of protein. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resolved a single protein-staining band that co-migrated with APase activity. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, glycosylated polypeptides of 57 and 55 kD were observed. The two polypeptides are immunologically closely related, since both proteins cross-reacted on immunoblots probed with rabbit anti-(Brassica nigra APase) immunoglobulin G. Immunoblotting studies revealed that the 55-kD subunit did not arise via proteolytic cleavage of the 57-kD subunit after tissue extraction. The native molecular mass was approximately 100 kD, suggesting that the holoenzyme could exist as either a homodimer or a heterodimer. The enzyme displayed a pH optimum of 5.8, was activated 40% by 4 mM Mg2+, and was potently inhibited by molybdate, vanadate, and ZnCl2. The final preparation displayed the highest activity and specificity constant with P-Tyr, but also dephosphorylated other phosphomonoesters including p-nitrophenylphosphate, O-phospho-L-serine, phosphoenolpyruvate, PPi, and ATP. Antibodies to P-Tyr were used to demonstrate that several endogenous phosphotyrosylated tuber polypeptides could serve as in vitro substrates for the purified APase. Although the precise physiological significance of the potato APase's substantial in vitro activity with P-Tyr remains obscure, the possibility that this APase may function to dephosphorylate certain protein-located P-Tyr residues in vivo is suggested.     

Improvement in the Histochemical Demonstration of Acid Phosphatase, β-Galactosidase and Nonspecific Esterase in Glycol Methacrylate Tissue Sections by Cold Temperature Embedding

A simple, cold-embedding method (on cracked ice at 2 C) is presented for the demonstration of acid phosphatase, β-galactosidase and nonspecific esterase in glycol methacrylate tissue sections.     

Fertility Preservation Options for Men and Women With Cancer

Approximately 0.2% of Americans aged 20 to 39 years are childhood cancer survivors. Advances in cancer detection and therapy have greatly improved survival rates for young cancer patients; however, treatment of childhood cancers can adversely impact reproductive function. Many cancer patients report a strong desire to be informed of existing options for fertility preservation and future reproduction prior to initiation of gonadotoxic cancer therapies, including surgery, chemotherapy, and radiotherapy. This article discusses, in detail, the effects of cancer treatment on fertility in men and women, and outlines both current and experimental methods of fertility preservation among cancer patients.Key words: Fertility preservation, Childhood cancer, Sperm cryopreservation, Testicular tissue cryopreservation, Spermatogonial stem cell cryopreservation, Embryo cryopreservation, Oocyte cryopreservation, Ovarian tissue cryopreservationIn 2014, an estimated 15,780 new cancer cases were diagnosed among children and adolescents younger than age 20 years, resulting in 1960 deaths. In addition, 1 in 285 children will be diagnosed with cancer before age 20, and approximately 0.2% of Americans aged 20 to 39 years are childhood cancer survivors.¹ Advances in cancer detection and therapy have greatly improved survival rates for young cancer patients; however, treatment of childhood cancers can adversely impact reproductive function (eg, men who survive childhood cancer are half as likely as their siblings to father a child).² Many cancer patients report a strong desire to be informed of existing options for fertility preservation and future reproduction.³ Therefore, the American Society of Clinical Oncology and the American Society for Reproductive Medicine recommend that consideration of fertility preservation be included prior to initiation of gonadotoxic cancer therapies, including surgery, chemotherapy, and radiotherapy.^4–6Infertility as a result of cancer treatment can be psycho logically upsetting for many patients,^3,7,8 and data suggest that those who pursued fertility preservation usually cope better with their cancer treatment.⁹ Infertile cancer survivors have an option to become parents through adoption or gamete donation, but most declare a preference for having a biological child.^3,10 Schover and colleagues³ found that 51% of newly diagnosed young male cancer patients reported a desire to have children in the future, and this rate increased to 77% for those who did not have children at the time of diagnosis. The desire to become a biological parent persists in male cancer survivors, as 70% reported wanting to father a child after chemotherapy treatment.⁹ A history of cancer treatment may be perceived by some to pose an increased risk to the health of future offspring; however, several studies have shown that male cancer survivors have not demonstrated an increased risk for having a child with birth defects or cancer.^11,12 Recently, a retrospective cohort study conducted in the United States showed no increased risk of malformations or premature birth in the offspring of male cancer survivors.¹³The optimal time for consideration of fertility preservation is before the initiation of any oncologic therapy that can affect gametogenesis; thus, it is critical that fertility preservation is discussed with all patients at the time of diagnosis and before treatment starts. Practitioners who provide care for cancer patients should be aware of the relationship between cancer treatment and infertility. Moreover, they need to be able to appropriately refer patients to a reproductive medicine specialist in a timely fashion for further counseling and fertility preservation. Although fertility concerns are paramount to young adults with cancer, many oncologists still do not routinely address these concerns.^3,14 In a survey of 200 young male cancer survivors who were primarily treated at a comprehensive cancer center, only 51% recalled being offered sperm cryopreservation prior to their cancer treatment.³ Further, it is important to recognize the psychologic stressors associated with a new cancer diagnosis and associated late effects of cancer treatment, such as infertility or early menopause. Findings from several studies support the importance of counseling patients regarding their risk for fertility issues and educating providers regarding the potential fertility preservation options that are available. For example, Babb and colleagues¹⁵ found that, at many institutions, this counseling is already taking place and there is a high rate of discussion with newly diagnosed patients regarding infertility.