Exploration of Methods for Preparation of Cattle Eyeball Paraffin Sections

[Objective] This study aimed to establish a new method for preparing paraffin sections of cattle eyebal s. [Method] The conventional method was used to prepare paraffin sections for cattle eyebal s in the control and a new method termed＆quot;opening a window on cornea and refixation＆quot; was used to prepare paraffin sections for cattle eyebal s in the treatment group. [Result] After the prepared specimens in the treatment group were fixed, it could be macroscopical y observed that retina and choroid were closely connected, with detachment occurring at a smal portion be-tween the two. According to the paraffin sections, it was microscopical y observed that the continuity of trabecular meshwork was intact, as wel as the continuity be-tween different layers of eyebal wal , without detachment between them, no retinal detachment, no shrinkage of each layer of tissue cells. [Conclusion] This study pro-vides a foundation for the basic research and pathological study of eyebal s.

小鼠整胚石蜡切片制备方法的改进

Objective:The morphology analysis of whole-mount mouse embryos was observed using an improved paraffin section technique.Methods:Mouse embryos of varying embryonic ages were collected and whole-mount embryo paraffin sections were prepared using PFA-intravenously injected fixation,prolonged dehydration,and paraffin embedding.Hematoxylin and eosin(H&E)staining and immunohistochemical staining were employed to evaluate the quality of sections,with different tissues being observed labeled by CD34.Results:Following a series of tissue processing and staining procedures,the structure of the whole-mount mouse embryo was well-preserved,and the staining was clear and easily distinguishable.Embryos of different embryonic ages were treated differently,yet the quality of tissue processing remained highly consistent.Conclusion:Tissue processing and staining have been significantly improved,allowing for the easy acquisition of whole-mount mouse embryos of different ages through simplified methods of tissue fixation and dehydration duration.The staining results are clear and stable,providing technical support for the study of mouse embryo development.

Paraffin section observation of flower bud differentiation of Chimonanthus praecox in Kunming and comparison of the differentiation processes in different regions,China

Chimonanthus praecox is an important ornamental plant and cut flower material in China.It blooms in the freezing winter and its flower emits charming fragrance.However,in different region the flowering time is variable.In order to understand the flowering mechanism of Ch.praecox in the winter,we studied the flower bud differentiation in Spring City-Kunming using paraffin sectioning method in the present study.Meanwhile we compared the differentiation process difference from different regions.It was found that the temperature is the key factor for its flower bud differentiation and blossom of Ch.praecox.In the process of bud differentiation,the temperature 20℃was the optimum for inducing changes from vegetative axillary buds to reproductive buds and subsequent morphological differentiation in Ch.praecox.Furthermore in the first three differentiation periods—tepal primordial stage,staminal primordial stage and pistil primordial stage,Kunming took the shortest time to finish the process due to very rapid temperature rise to 20℃,whereas,in Zhengzhou the time for these differentiations was the longest,which may be caused by the slow temperature rise.After May,the high temperature stress forced the flower buds into the first long dormant period in all regions except Kunming.In Kunming,the average temperature was only 20–25℃,so the flower bud continued to differentiate.In all regions,Kunming is the first to complete whole flower bud differentiation even on the early August,and started the second dormancy very early but very long.In the other regions,the plants went through a shorter dormancy and the low temperature broke the dormancy rapidly.Contrarily the plants of Kunming spent a longer period for the low temperature.Thus,the low temperature less than 10℃is a key factor to breaking the second dormancy.Surely the regular effects of temperature on flower bud differentiation and blossom is very helpful for florescence regulation of Ch.praecox.

Preparation of Paraffin Section of Pinus sylvestris Needles

[Objective] Taking Pinus sylvestris needles as materials, a set of test con-ditions suitable for the preparation of paraffin sections of Pinus sylvestris needles was determined. [Method] Based on the traditional method for paraffin sections preparation, steps including fixation, dehydration, adhering to slides and staining were investigated taking the structural characteristics of pine needles into considera-tion. [Result] 70% ethanol was used in the FAA fixative; before affixed on slides, the cut sections were first expanded in a 40 ℃ water bath and taken out by using Su-perfrost Plus slides, fol owed by drying in a 35 ℃ oven for 24 h; pine needles were cut into 8 μm thick sections; staining was achieved by immersion in eosin for 5 min and in hematoxylin for 1 min. [Conclusion] The conditions obtained above can en-hance the effect of fixation and dehydration; adhering to slides is easy to operate and the sections are not easy to drop; the dyeing effect is relatively preferable.

A Preliminary Study on Fertilization Biology between Epinephelus coioides(♀) and Epinephelus lanceolatus(♂)

Crossbreeding between Epinephelas coioides （ ♀ ） and Epinephelus lanceolatus （♂） was conducted by artificial insemination. Fertilized eggs were col- lected at 0, 30, 90 s, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min post-insemination respectively, which were fixed by Smith＇s solution, embedded by paraffins and stained by H.E. According to the characteristics of Epinephelus coioides eggs, tissue section method was modified. The results showed that the sperms of Epinephelus lanceolatus rapidly entered the eggs of Epinephelus coioides at 30 s - 1 rain post-insemination. Observation results of tissue sections showed that mature eggs of Epinephelus coioides remained at metaphase of secondary maturation division. The eggs were activated after sperm penetration into the egg. With the develop- ment of secondary maturation division, at 2 rain post-insemination, eggs reached the metaphase of secondary maturation division ; at 3 - 6 rain post-insemination, sperm asters appeared; at 5 min post-insemination, eggs extruded secondary polar body; at 7 -15 rain post-insemination, male pronucleus and female pronucleus moved closer to each other and fused finally, forming a clear junction line; subsequently, zygote nucleus formed and karyotheca became faint; at 15 min pest-insem- ination, first karvokinetic division was develoued.

Immunofluorescence on paraffin embedded renal biopsies:Experience of a tertiary care center with review of literature

AIMTo describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the po-tential pitfalls with an in depth review of literature.METHODSImmunofluorescence is integral to diagnostic renal pa-thology. Immunofluorescence on paraffin embedded renal biopsies （IF-P） after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffn embedded renal biopsy material was standardized and applied prospectively in cases where immunofuorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofuorescence was performed.RESULTSOver the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases （7.7%） and was interpretable with optimal digestion in 214 cases （6.8%）. It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Non-diagnostic IF-P was found in membranous nephropathy （2 of 11 cases）, membranoproliferative glomerulonephritis （2 of 32 cases）, lupus nephritis （1 of 25 cases）, post infectious glomerulonephritis （1 of 11 cases） and chronic glomerulonephritis （3 of 8 cases）. Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining（1＋） for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue. CONCLUSIONAs a “salvage” technique, immunofuorescence on paraffn embedded renal biopsies is of great diagnostic utility, however not without pitfalls.

Cytological Observation of Microsporogenesis in Male-Sterile Lines of Chinese Pink(Dianthus chinensis L.)

This study addressed the differences in microsporogenesis between male sterile and fertile lines of Chinese pink. The microsporogenesis processes of male sterile and fertile lines were histologically examined in squashed pollen grains and in paraffin embedded sections. A stable male-sterile line （H-37B） was obtained following six generations of inbreeding in a self-fertile line, followed by two generations of backcrossing. In the corresponding fertile line, development of the mature pollen grains was followed through the initiation of the sporogenous cell, microsporocyte formation, and the tetrad developmental period. In the male-sterile line, abortion of the developing pollen grains was observed to take place at various stages, namely, sporogenous cell growth, mother cell meiosis, and tetrad transformation to the uninuclear state. The pollen grains of the fertile line were spheroid, turgid, and viable. By contrast, the male-sterile line produced pollen that was irregular in shape, empty, and nonviable. The abortion of the microspore in the male-sterile line appeared to relate to abnormal growth of the tapetum layer.

Observation on Flower Bud Differentiation of Crape Myrtle in Red Soil Environment

Flower bud differentiation is a key component of plant blooming biology and understanding how it works is vital for flowering regulation and plant genetic breeding,increasing the number and quality of flowering.Red soil is the most widely covered soil type in the world,and it is also the most suitable soil type for crape myrtle planting.The flower buds of crape myrtle(Lagerstroemia indica)planted in red soil were employed as experimental materials in this study,and the distinct periods of differentiation were identified using stereomicroscopy and paraffin sectioning.We optimized the steps of dehydration,transparency,embedding,sectioning and staining when employing paraffin sections.When seen under a microscope,this optimization can make the cell structure of paraffin sections obvious,the tissue structure complete,and the staining clear and natural.The flower bud differentiation process is divided into 7 periods based on anatomical observations of the external morphology and internal structure during flower bud differentiation:undifferentiated period,start of differentiation period,inflorescence differentiation period,calyx differentiation period,petal differentiation period,stamen differentiation period,and pistil differentiation period.The differentiation time is concentrated from the end of May to mid-June.Crape myrtle flower bud differentiation is a complicated process,and the specific regulatory mechanism and affecting elements need to be investigated further.

Production Method for Paraffin Section of Invasive Species of Bemisia tabaci

[Objective] The purpose was to introduce a preparation method for paraffin section of Bemisia tabaci, so as to lay the foundation for the studies on changes of organizational structure of B. tabaci. [ Method] The technique of paraffin section and the method of H-E staining were adopted to study the organizational structure of B. tabaci. The slices were examined and photographed under fluorescence microscope. [ Result] The coloring of H-E staining cells was good. Under 400 x conditions, the fat body, compound eyes, nucleus and muscle of B. tabaci were clear. [ Conclusion] The production method for paraffin section of B. tabaci was reliable, and the quality of slices was high.

Improving sample preparation to investigate lignin intensity of xylem at the cellular level by confocal Raman microspectroscopy of Populus tomentosa

Confocal Raman microspectroscopy(CRM)is an important tool for analyzing the compositional distribution of cell walls in situ.In this study,we improved the sample preparation method using paraffin-embedded sections combined with hexane dewaxing to obtain high resolution Raman images.We determined that the cell wall components of fiber cells were different from those of ray cells and vessel cells in the xylem of Populus tomentosa.Acetyl bromide and CRM methods produced similar trends when the difference in lignin intensity in the xylem region was compared between transgenic PtrLac4 and wild-type P.tomentosa.However,CRM proved more useful to analyze the lignin distribution in each cell type and distinguished the detailed difference in lignin intensity at the cellular level.Thus,CRM proved to be a useful in situ method to rapidly analyze the spatial variation of lignin content in the xylem of woody plants.

Effects of Solvents on Extraction of Effective Components from Cell Wall Tissues of Chinese Traditional Medicines

The effects of different Chinese traditional medicines were solvents on the extraction of effective studied by the histochemical methods, components from the cell wall tissues of such as the bare-handed section, swelling ratio, paraffin section, IR spectrum and cell wall component analysis. The results show that the ethanol-alkali solvent could increase the swelling ratio as well as the swelling speed. The effective components of cell wall tissues extracted by ethanol-alkali solvent become loose shown by the paraffin section. According to the IR spectrum analysis and the results of cell wall tissue component analysis, it was found that the ethanol-alkali solvent could decrease the contents of pectin and hemicellulose in the cell wall to make the wall broken, and therefore the effective components can be extracted easily by the solvent and the extraction rate is increased.

Interrelationships between leaf heat conductivity and tissue structures of different varieties of Populus tomentosa Carr

Plant heat conductivity largely depends on tissue structure. Different structures lead to different heat conductivity. As well, water transfer also plays a very important role in heat transfer in plants. We have studied leaf heat conductivity and tissue structure of 3- and 30-year-old Populus tomentosa Carr. trees using infrared thermal imaging, steady state heat conductivity surveys and paraffin section and investigated the relationship between leaf heat conductivity, tissue structure and water content of leaves. The results show that the temperature on leaf surfaces among the various varieties of trees was almost the same. Leaf heat conductivity, temperature and water content of leaves are positively correlated. The thicker the leaf tissue structures, the larger the heat resistance. That is, the tighter the cells and the smaller the interspaces, the smaller the heat conductivity, which is not conducive for heat transfer.

Comparison on Morphology and Micromorphology of Paris polyphylla Sm. var. polyphylla with Different Year Numbers

[Objectives] The research aimed to identify Paris polyphylla with different growth year numbers and evaluate its resource quality.[Methods]Comparative research on P. polyphylla with different growth year numbers was conducted by using morphological identification and microscopic identification method. [Results] By contrasting original plant morphology,transverse sections of rhizome and fibrous root,powder,it displayed that microstructure of P. polyphylla had regularity difference in cell shape and size,duct quantity,trait and size,size and distribution of calcium oxalate cluster crystal. [Conclusions] The microstructure change of P. polyphylla was related to the number of its growth year,which could provide the reference for identifying the growth year number of P. polyphylla and evaluating its resource quality.

RAPID DETECTION OF ONCOGENE AMPLIFICATION IN PARAFFIN SECTIONS OF BREAST CARCINOMAS USING QUANTITATIVE DIFFERENTIAL PCR AND FLUORESCENT DNA TECHNOLOGY

Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognostic information for the individual patient. Standard analytical approaches using Southern blot and slot blot require a large amount of good