Paraffin sectionHistologyThe most widely used method in conventional production technology.Paraffin section is not only used for observationnormal cells Organizationalmorphological structure , alsoPathologyandforensic medicineIt is the main method used by other disciplines to study, observe and judge the morphological changes of cell and tissue, and has also been widely used in the research of many other disciplines.
Chinese name
Paraffin section
Foreign name
paraffin section
Application
Observe the morphology and structure of normal cell tissue
Paraffin sectionIt is the most widely used method in conventional histological sectioning technology.Paraffin section is not only used for observationnormal cells Organizationalmorphological structure , alsoPathologyandforensic medicineIt is the main method used by other disciplines to study, observe and judge the morphological changes of cell and tissue, and has also been widely used in the research of many other disciplines.In teaching, most of the sectioned specimens observed under light microscope are prepared by paraffin sectioning.Living cells or tissues are mostly colorless and transparent, and there is no contrast between various tissues and various structures in cells, which is difficult to distinguish clearly under general light microscope;After leaving the body, the tissue will soon die and become corrupted, losing its original normal structure. Therefore, the tissue should be fixed, paraffin embedded, sectioned and stained to avoid cell death, and its morphology and structure can be clearly identified.
Basic technology
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The paraffin sectioning method includes material extraction, fixation, washing and dehydration, transparencySoaking wax, embedding, slicing and pasting, dewaxing, dyeing, dehydration, transparencyCoverAnd so on.Generally, it takes several days for tissues to be taken and fixed to be made into slide specimens on the cover, but the specimens can be stored and used for a long time and are permanentMicroscopic slide specimen。
Material acquisition
The material source and position shall be selected according to the requirements.For example, onion root tips are often selected for plant cell mitosis, so cell division is fast and easy to cut;The borders of pig liver lobules are clear;The inner ear of guinea pig is easy to locate and peel off the cochlea.The material must be fresh. If it is kept for too long, it will cause protein degradation and denaturation, which will lead to cell autolysis and bacteria breeding, but cannot reflect the morphological structure of living tissue.
fixed
Immerse the fresh material cut into small pieces with appropriate chemical liquid - stationary liquid, rapidly coagulate or precipitate the material components in cells and tissues, terminate all metabolic processes of cells, prevent cell autolysis orOrganizational changesAnd keep its living structure as much as possible.The fixation can harden the tissue, which is conducive to the sectioning, and also has the role of media leaching, which is conducive to tissue coloring.There are many kinds of fixatives, such as the degree of hardening and shrinkage of tissues, and the proteinFat, sugars and other substances have different functions.For example, pure alcohol can be fixedHepatoseIt can dissolve fat,formaldehydeIt can fix general tissues, but dissolve liver sugar and pigment.The fixative can be divided into single fixative and mixed fixative.The former has formaldehyde（acetaldehydeFormalin), alcoholacetic acidorGlacial acetic acid、Mercuric chloride、Osmic acid（Osmium tetroxide）、potassium dichromateAs well as picric acid, a single fixative cannot fix all components in cells;Mixed fixatives can complement each other. Common mixed fixatives includeBouin's solutionZenker's solution, FAA solution, Carnoy's solution, SuSa solution (see relevant technical books for the formula).Therefore, appropriate fixative should be selected according to the content to be displayed.10% formalin (4% formaldehyde) or 10%phosphoric acidBuffer formalin isPathological sectionThe fixed liquid used routinely is not only suitable for conventionalHematoxylin-Eosin(HE) staining can also be used for section dyeing of other technologies related to histology.The amount of fixative is usually about 20 times that of the material block. The fixation time depends on the size and density of the material block and the penetration speed of the fixative, which can range from 1 hour to several days, usually from 1 hour to 24 hours.
Washing and dewatering
After fixation, the fixative solution and crystal precipitation left in the tissue should be removed, otherwise, the dyeing effect in the later stage will be affected.This step is called washing mostlyFlushing with running water；If the fixative solution containing picric acid is used for fixation, it needs to be soaked with alcohol for many times;Use alcohol or alcoholMixed liquidFixed tissue can be dehydrated directly without washing.The fixed or washed tissues are full of water. If the water is not removed, the subsequent transparent, wax dipping and embedding treatment cannot be carried out. The reason is thatTransparent agentMost of them are benzene. Benzene and paraffin cannot be mixed withaqueous phaseDissolve, moisture cannot be removed, and benzene cannot be immersed.Alcohol is commonly usedDehydrating agent, it can not onlyPhase mixingAnd can be mixed with transparent agent.In order to reduce the sharp shrinkage of tissue materials, it should be carried out in the order of increasing from low concentration to high concentration, usually starting from 30% or 50% alcohol, and going through 70%, 85%, 95% to pure alcohol（Anhydrous ethanol）The time of each time is 1~several hours. If the dehydration at all levels cannot be carried out in time, the material can be stored in 70% alcohol. Because high concentration alcohol is easy to shrink and harden the tissue, it should not be treated for too long.N-butanol、Tert butyl alcoholAcetone and dioxane can also be used as dehydrating agents.
transparent
Pure alcohol cannot be miscible with paraffin, and it is also necessary to replace the alcohol in the tissue with a solvent that can be miscible with alcohol and paraffin.When the material block is dipped in this kind of media, it becomes transparent. This liquid is called transparent agent, and the impregnation process of transparent agent is called transparent.Common transparent agents arexylene, benzenechloroform, n-butanol, etc. Various transparent agents are solvents for paraffin.Usually, the tissue is soaked for 1~2 hours in a mixture of pure alcohol and half of the transparent agent, and then transferred to the pure transparent agent for immersion.HyalineImmersion timeIt depends on the size of the tissue material block and whether it belongs to the capsule or the parenchymal organ.If the transparent time is too short, the transparency is not complete, and paraffin is difficult to immerse into the tissue;If the transparent time is too long, the tissue will harden and become brittleCut outFull sectioning, up to several hours.
Waxing and embedding
Replace the transparent agent with paraffin, so that paraffin can be immersed into the tissue and play a supporting role.Generally, the tissue material block is immersed in the same amount of mixed solution of melted paraffin and xylene for 1~2 hours, and then moved into two melted paraffin solutions successively for about 3 hours. The immersion wax should be higher thanMelting point of paraffinIt is carried out in a warm box at about 3 ℃ to facilitate paraffin immersion into tissues.The tissue material block after wax dipping is placed in a container containing wax liquid (placed in the wax). When the surface of the wax liquid solidifies, it is quickly put into cold water for cooling, which is to make a wax block containing tissue blocks.Containers can be folded into paper boxes or metal encased boxes with bright and thick paper.If there are many embedded tissue blocks, they should be numbered to avoid errors.After the wax is melted, it should be filtered in the wax box before use, so as not to affect the quality of the slice due to impurities and may damage itMicrotome knife。Generally, paraffin wax has a melting point of 56~58 ℃ or 60~62 ℃, depending on the season and operationambient temperatureTo choose.
section
The embedded wax block shall be trimmed into regular shape with a bladeQuadrangular frustum, quickly attach the bottom with a little hot wax liquid to the small wooden block, and clamp itRotary microtomeMake the cut surface of the wax block parallel to the cutting edge of the slicing knife and tighten it.The sharpness of the slicing knife and the proper hardness of the wax block will directly affect the quality of the slice. Hot water or cold water can be used to properly change the hardness of the wax block.Generally, the thickness of the slice is 4~7 microns. Cut out one piece of wax tape after another, and gently place it on the paper with a brush.
Patch and toast
Flatten with adhesiveWax flakeAttach toSlideTo prevent the two from slipping off in the subsequent steps of dewaxing, hydration and dyeing.Adhesive is proteinglycerol。First, apply a thin layer of protein glycerin on the clean slide, and then apply a certain length of wax tape（Continuous sectioning）Or use a blade to break it into a single wax slice, flatten it in warm water (about 45 ℃), and then put it on the glass slice, or directly drop two dropsdistilled waterPut the wax slice on the glass slide, put it on the water drop, slightly warm it to spread the wax slice, and finally use filter paperAspirateThe excess moisture can be dried in a 45 ℃ oven or 37 ℃ oven, but the time needs to be extended appropriately.
Section dewaxing and hydration
The dried sections need to be dewaxed and hydrated before dyeing in water-soluble dye solution.Dewaxing with xylene, then step by step through pure alcohol and gradient alcohol until distilled water.If the dye is prepared in alcohol, the slice can be dyed when it is moved to a concentration similar to that of alcohol.
dyeing
Paraffin section
The purpose of staining is to make different structures in the cell tissue present different colors for easy observation.Unstained cell tissueRefractive indexSimilar, difficult to identify.Staining can show differentOrganelleandInclusionAnd different types of cell tissues.StainThere are many kinds of dyes. Different dyes and dyeing methods should be used according to the observation requirements and research contents, and proper fixatives should be selected to obtain satisfactory results.Classic Hematoxylin and Ehong（Eosin, Eosin) staining is a method ofPathological sectionRoutine staining of specimensHE staining。After HE staining, the nucleus is dyed purple blue by hematoxylin, most of which arecytoplasmAnd non cellular components were dyed pink by eosin.Because hematoxylin iscationThe dye solution is alkaline, and inside the nucleusChromatinAnd cytosolic ribosomesAffinity, saidBasophil；WithanionThe dye solution prepared with eosin is acidic, and its affinity for this dye is calledAcidophilic。Sometimes differentorganization structureIt also needs to be displayed with special dyes and dyeing methods, called special dyeing.Some cell tissuessilver nitrateAfter soakingSilver ionReduction to metallic silver orSilver granuleIt attaches to the cell tissue and is brown and black, which is called argentaphilic. Some cell tissues themselves cannot reduce silver ions of silver nitrate to metallic silverreducing agentTo reduce silver ionsArgyrophilic。
Slice dehydration, transparency and sealing
The stained sections cannot be observed under the microscope, but need to be dehydrated by gradient alcohol, and the time in 95% and pure alcohol can be appropriately prolonged to ensure complete dehydration;If the dye solution is prepared with alcohol, the time in alcohol should be shortened to avoid decolorization.After the xylene is transparent, quickly wipe off the excess liquid around the material,Dropwise additionAppropriate amount (1-2 drops)neutral balsam , and then cleanCover slideTilt it down to avoid bubbles, and make permanent slide specimens after sealing, which can be observed repeatedly for a long time under the light microscope.Note that some dyes need to be produced by specific manufacturers.According to various dyeing methods, tissue types and slice thickness, mastering the appropriate dyeing time can achieve better dyeing effect.
There are many procedures and links for paraffin sectioning. It takes several days to complete a cycle, but the slices can be preserved for a long time for teaching, scientific research andpathologic diagnosis And reexamination, and the wax block can be used for retrospective study of other items.Some detailed links have been simplified or shortened during routine pathological preparationprocessing time To meet clinical needs (can be shortened to 2 days).althoughfrozen section It is much faster than paraffin section, but the morphological structure shown is not as good as the latter, soPathologistFinally, accurate diagnosis should be made according to paraffin sections.In recent years, microwave technology has been used in routine pathological preparation, which has greatly shortened the preparation process and has no impact on the morphology and structure.Microwave is a kind of short wavelength, but high frequencyHigh frequency electromagnetic wave[Wavelength: 1 m~1 mm, frequency: 300 MHz（MHz）~300 GHz（GHz）]。Organization Channelmicrowave radiationPost accelerationHigh speed movement of molecules inside the organization to accelerate the transportation of liquid, increase dispersion, penetration andExchange efficiencySo as to accelerate the fixation, dehydration, transparency, embedding and dyeing of tissues.For example, routine formalin fixation takes several hours to one day, and can cause tissue contraction and destruction of some antigen components to varying degrees. Microwave fixation only takes one to two minutes, and can reduce the loss and damage of antigen.It is very important to select the appropriate grade (power), radiation time and temperature.The application of microwave technology is still in its infancy in ChinaTechnology applicationThe link needs further exploration.
Paraffin sectioning is not only a classic method, but also the most basic method. It combines with other new technical methods to expand the traditional old technologyScope of applicationIt has opened up many new fields and added many new research and observation contents.With the advent and use of new instruments and new research technologies, the observation and research of histology has gone from simple morphological structure to qualitative observation of various components, and from qualitative to quantitative measurement, so that the morphology, function and metabolism of cell and tissueTriple combinationSo as to achieve qualitative reliability, accurate positioning and quantitative measurability.
Technology integration
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Organizational production technology andimmunologyTechnical combination compositionImmune tissue(Cellular) chemical technology, using antigens andantibodyOfSpecific bindingPrinciple, detect the polypeptide andproteinQualitative and positional observation of macromolecular substances.Either wayImmunohistochemistryThe technology includes antibody preparation, tissue material treatment and preparationslideSpecimens andImmunostaining。frozen section The procedure is simple, and the antigen activity is less lost during the production process, butHistiocytePoor morphology;The paraffin section has many steps, and the antigen activity in the section is reduced, but the histocyte morphology is clear. It is one of the routine methods for preparing sections in immunohistochemistry.commonlyParaffin embeddingTissue section of for detectionCytoplasmOr intranuclear antigen, not suitable forsurface antigen Staining, someone soaked fresh tissue in coldPhosphate bufferWithin 48 hours, and then fixed at 96%ethanolOr othersStationary fluidThe fixed tissue section can be used for surface antigen staining.Ethanolacetoneetc.FixativeThe antigenic damage is light, but the structure preservation is poor.The most commonly used fixatives are neutral and bufferformalin, it can cross bind with protein to block antigenImmunostainingBefore slicingproteaseDigestion to expose antigen sites and enhance antigen response.The commonly used immunohistochemical staining on paraffin sections includesEnzyme histochemistryPAP method in technology (non markingPeroxidase-Anti peroxidase method) and affinityImmunohistochemistryTechnicalABC method(avidin biotin peroxidase complex technology), whichSpecificityStrongsusceptibilityHigh.Use twoDyeing methodThe first antibody (variousantiserum）Incubation;PremenstrualSecond antibodyOr biotin labeled second antibody incubation;After incubation with PAP complex or ABC complex, DAB（Diaminobenzidine）-The H2O2 solution is brownish yellow and precipitated.Conventional re dyeing, dehydration, transparency, and sealingPermanentThe slide specimen can be used to detect the components that are difficult to be displayed by conventional or special staining methods and their precise positioning under light microscopefundamental researchAnd clinicopathological study and diagnosis.Microwave used for immunohistochemical staining of paraffin embedded sections not only simplifies the steps and saves time, but also can promoteImmunostainingeffect.
Paraffin embedded tissueFlow cytometryDNA content analysis is to compare paraffin embedded tissue sections withFlow cytometry（flow cytometry，FCM）It is used in combination to measure DNA content and ploidy analysis. This combination is the combination of flow cytometry in clinical application, especially intumourThe research has opened up new research approaches.FCM is laser, electron and electroncomputer. Fluid injection technologycomprehensive developmentApplication is a technology for rapid and quantitative analysis of cells, which requires that the detected cells be in a suspended state.The size, volume, DNA contentDNA synthesisRateRNAContent, surface antigen, chromosome, etc.Due to the technology of sample preparation, many flow analysis data in the past were limited to fresh tissue samples. Hedley et al. first reported in 1983 that FCM analysis paraffin embedded tissue sections were used to prepare dispersed cell suspensions to detect DNA content. A sufficient number of single cells could be obtained from tissue sections, andTissue segregationThe single cells obtained are very similar in morphology and DNA content group diagram.Research in this field is also being carried out gradually in China.Because this method can be taken on the basis of pathological tissue observation or diagnosis, it is more accurate than biopsy or surgical resectionRetrospective studyanalysis;The DNA detection is fast and the data is objective and reliableprognosisForecast andTherapeutic responseHas important value;Cell suspension can be made in batches and tested on the computer in batches by using the past samples, avoiding the impact of different instrument commissioning states. The paraffin embedded blockSave timeThe length of has little effect on the results.
Conventional fixation (mostly formalin) and paraffin embedded tissue blocks can be used forFlow cytometry, but withPicric acidOr mercury fixative should not be used.The slice thickness should be 30~50 μ m.sectiondewaxingIt should be clean and thorough, otherwise the prepared cell suspension contains many fragments, which will affect the determination.Used after gradient alcohol hydrationPepsinorTrypsinDigestion: after digestion, microscopic examination showed a single scattered state;Because formalin fixation can make DNA andNucleoproteinproduceCovalent crosslinking, affecting DNA anddyestuffChemical quantitative combination of, resulting influorescence intensityDecrease, protease can destroy its connection, improve the quality of the measured recipe, and digest time to disperse cells andCell debrisAs little as possible, use 100 mesh nylon screen to filter the undigested tissue slice and centrifugate itsupernatantAfter that, add cold 70% alcohol to fix it, and put it into the 4 ℃ refrigerator for standby.Dye before use.DNA specificFluorescent dyeMainly include: (1)Propidium iodide（PI）；（2）Ethidium bromide（EB）；(3) 4, 6-IIAmidino-IIphenylindole（DAPI）。FCM detection paraffin embeddingHistiocyteThe DNA content can be used asmorphologyBased on, multi parameterComprehensive diagnosisAn important new technique in.Due to the high price of instruments and equipment and the fact that many links of sample preparation may affect the measured data, the widespread clinical use is limited.
Paraffin section specimenIt is the basis of morphometric technology. morphometric technology is a new quantitative technology developed in recent yearsDetection technology, usingFull automatic image analyzerThe quantity, volume, length andSurface areaWaitingimage dataMathematically processed toHistiocyteAnd its structural components, such asmitochondrionNumberEndoplasmic reticulum、nucleusandCytoplasmAreaIslets of langerhansThe number of cells and the number of cellsRenal corpuscleQuantity and volume ratio of andfeulgen staining Determination of nuclear DNAIn situ quantificationWaitHistologyandcytologyThe research on the change from morphological and qualitative observation to morphological quantification.Morphometric measurements are taken from paraffin tissue sections orsmearAnd various structures on the light microscope photos and electron microscope photos of the tissue to obtain two-dimensional image datasoftware program And get valuable structural parameters.Such dataaccuracyHighobjectivityStrongRepeatabilityGood, can reduce or make up for the observersubjectivityDifferences.Morphometry has been applied in clinical practicepathologic diagnosis Work, including non tumor lesions and tumor lesions.Domestic research on non neoplastic lesions has involvedatherosclerosis、cirrhosis、Hypertrophy of prostate、Cretinism、pancreatitisandVaricoceleEtc;The study of tumor lesions is based on morphometryclinical research Focus on the digestive tract, liver, bladderbreastAnd lung and other organs.In addition, the combination of morphometric parameters and functional change indicators in the process of disease occurrence is conducive to exploring thePathogenesis；Morphometric parameters also have reference value for the estimation of therapeutic effect and prognosis of tumor lesions.
In the study of morphometry, the first step is to study the samplesquality requirementHigh, such as shrinkage, expansion and extrusion of tissue during sectioning, slice thickness and direction, especially sliceDyeing technologyStaining is to make each component of the tissue or cell dyed into different colors to facilitate identification and improve the image of the component to be measuredMeasurabilityThe expressiveness of its internal components makes the color and brightness of the component to be measured clearly distinguish from its background.Conventional dyeingSpecial dyeingas well asImmunohistochemistryDyeing, which can be applied to different colored components andreactantQuantitative morphological determination;Using immunohistochemistry andIn situ hybridizationThe lesion was stained and then quantitatively measured.The second is to find outfeature structureParameter orQuantitative indicatorsYou can first detect various measurable parameters of tissues or cells or design some parameters according to the morphological characteristics of the components to be measured, and then use statistical methods to screen out useful parameters for quantitative determination.Due to the high cost of instrumentsexperimental study At this stage, there are still some difficulties in the wide clinical application.Integration of morphometric technology with conventional technology and other new technologiesresearch methodIt has good application value and effect.
With the advent of various new instruments and the continuous establishment and use of new technologies and methods, paraffin sectioning technology has gradually expanded and infiltrated into many new fields asBasic technologyProvide effective experiments or use samples.Paraffin embedded tissue sections can also be used for cellsIn situ nucleic acid molecular hybridizationMedium, which can be used forDNA moleculePositioning, content analysis or observationgene expression（mRNA）Level;polymerase chain reaction (PCR) technology can be used for DNA analysis of fixed and paraffin embedded tissues, making the research enter the molecular level, but these technologies cannot be used routinely in the short term.
