CN103940648A - Preparation method for gill tissue paraffin section - Google Patents
Preparation method for gill tissue paraffin section Download PDFInfo
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- CN103940648A CN103940648A CN201410133076.1A CN201410133076A CN103940648A CN 103940648 A CN103940648 A CN 103940648A CN 201410133076 A CN201410133076 A CN 201410133076A CN 103940648 A CN103940648 A CN 103940648A
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Abstract
The invention discloses a preparation method for a gill tissue paraffin section. The preparation method comprises the following steps: fixing, decalcifying, dehydrating, transparentizing, carrying out paraffin permeation, embedding, slicing, sticking sections, expanding the sections, de-waxing and rehydrating, staining, re-staining, sealing and the like. Compared with an existing paraffin section manufacturing method, an operation process of dehydrating, transparentizing and immersing by wax is improved; the preparation fixing and tissue wax immersing effects of gill tissue paraffin are improved; a slicing problem when the gill tissue paraffin section is prepared is improved; the structure definition of the gill tissue section is greatly improved; a plurality of problems in a gill tissue manufacturing process in the prior art are solved. The preparation method is good for antigen positioning of immunocytochemical staining, so that when experiments including in-situ hybridization, immunohistochemistry, immunofluorescence and the like are carried out on the gill tissue paraffin section, tissue distribution and cell positioning of some genes and proteins can be displayed, and further feasible conditions are provided for carrying out gill research on levels of cells, genes and proteins.
Description
Technical field
The invention belongs to microstructure section field, be specifically related to the preparation method of a kind of fish gill tissue paraffin section.
Background technology
Paraffin section (paraffin section) is the method for widespread use the most in the conventional tabletting technology of histology.Paraffin section is not only for observing the morphosis of normal cell tissue, also be the subjects such as pathology and medical jurisprudence in order to research, observe and the main method of the metamorphosis of judgement cell tissue, and be quite widely used in the research of other many ambits.In teaching, light Microscopic observation section preparation majority is prepared by paraffin method.The cell or tissue of living mostly is water white transparency, all lacks contrast between various tissues and in cell between various structures, is difficult for clear distinguishing under general light microscopic; Tissue leaves after body will soon be dead and to produce tissue corrupt, loses original normal configuration, and therefore, tissue will be through steps such as fixing, paraffin embedding, section and dyeing in order to avoid cell tissue be dead, and its morphosis of the clear identification of energy.Its basic process comprises: fixing, dehydration, transparent, ooze a plurality of steps such as wax, embedding, section, exhibition sheet, glutinous sheet, roasting sheet, dewaxing rehydration, dyeing and sealing.
The fish gill of bony fish has five pairs of visceral arch, and first four pairs have the gill lamella that 2 row are elongated, and it connects by film between the gill substantially, and the visceral arch front side row of many fingerlings are being given birth to gill raker.There are many semicircular secondary gill lamellas the both sides of the gill lamella, consist of the squamous epithelium of individual layer above it epithelial cell, and by between columnar cell across, many capillaries of parallel distribution.Due to the singularity of fish gill tissue structure, adopt traditional paraffin section method for making, be difficult to obtain desirable effect, not only structural fuzzy, and nucleus and cytoplasm dyeing contrast are not obvious, lack unity and coherence, painted bad, cause the making failure of tissue specimen.Therefore, how existing paraffin section preparation method being improved, solve the problem existing in fish gill tissue section preparation process, is this area problem demanding prompt solution.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, the preparation method of a kind of fish gill tissue section is provided, this preparation method can guarantee fish gill tissue and eucaryotic cell structure complete, position is relatively fixing, and simple, efficient, be applicable to the rapid large-scale preparation of fish gill tissue section.
For achieving the above object, this invention has adopted following technical scheme.
The method for making of fish of the present invention gill tissue paraffin section, comprises that step is as follows:
(1) fritter that fixedly Jiang Yu gill tissue is cut into 3 mm * 2, mm * 3 mm is soaked in Bouin ' s solution and is fixed, and fixes 24 h under room temperature condition, during change Bouin ' s solution 2 ~ 3 times;
(2) the material flowing water that (1) is processed in decalcification rinses 24 h, then immerses in Perenyi ' s decalcifying Fluid, keeps 5 d under room temperature condition;
(3) dehydration is immersed ascending gradient ethanol dehydration successively by the material after (2) processing, step is successively: 60% alcohol immersion 0.5 h, 70% alcohol immersion 0.5 h, 80% alcohol immersion 0.5 h, 90% alcohol immersion 1 h, 95% alcohol immersion 1 h, 95% alcohol immersion 2 h, soaked in absolute ethyl alcohol 40 min, soaked in absolute ethyl alcohol 40 min;
(4) transparently (3) are handled well to material immerse successively that dimethylbenzene soaks 15 min, dimethylbenzene soaks 15 min;
(5) paraffin infiltration is immersed dimethylbenzene and paraffin and in the solution of 1:1, is soaked 30 min by volume handle material well through (4), then transfers in the paraffin of 56 ℃ and permeates 4 h;
(6) embedding is converted into small paper box by kraft, and the paraffin first dissolving heating is poured in carton, then rapidly will through (5) handle well material tangent plane down, the gill lamella and tangent plane laid parallel in carton, cooling forming;
(7) section, by (6) embedded paraffin mass through over-segmentation, finishing and set, is then cut into continuous wax band through microtome by paraffin mass, and wax belt shape is rectangle, and upper and lower two long limits are parallel, and slice thickness is 5 ~ 6 μ m;
(8) the wax band that exhibition sheet and glutinous sheet cut (7) is cut apart the fragment that is suitable, be placed on the distillation water surface of 50 ~ 55 ℃, while observing the abundant expansion of section, with the microslide that scribbles glutinous sheet liquid, section is fished for rapidly, section is controlled to the optimal viewing position of microslide, slide is placed on to roasting sheet 12 h of constant temperature oven of 40 ℃, the labelled or mark of section after drying, is put in slide storage case;
(9) dewaxing rehydration is cut into slices and is dewaxed after (8) are processed, be immersed in successively twice of dimethylbenzene, be 5 min at every turn, then carry out descending gradient ethanol rehydration, be immersed in successively absolute ethyl alcohol 5 min, absolute ethyl alcohol 5 min, 95% ethanol 3 min, 70% ethanol 5 min, then with clear water, rinse 5 min, distilled water flushing 1 min;
(10) dyeing is put into haematine 15 min that dye by the section of processing through (8), with tap water, rinses after 1s, puts into percent by volume and is 1% ethanol solution hydrochloride and break up the 1s that fades;
(11) redye the section that (9) are processed and put into after tap water flowing water rinsing 10 min, distilled water flushing 1s, through Yihong ethanolic solution, redye 1 min, then distilled water rinsing 1-5 s; Finally by crossing 75% ethanol, swing and wash 1-5 s, 95% alcohol flushing 1 min, absolute ethyl alcohol and rinse 1 min, absolute ethyl alcohol and rinse that 1 min, dimethylbenzene rinse 3 min, dimethylbenzene rinses 3 min;
(12) be locked in after the Canadian resin that in the section of processing through (10), a 2-3 drips, rapidly cover glass carefully covered, can not produce bubble, if there is bubble to squeeze out, then that mounting is at room temperature natural drying.
Described Bouin ' s immobile liquid is formulated by volume by following reagent: 15 parts of saturated picric acid, 5 parts of formalin, 1 part, glacial acetic acid.
Described Perenyi ' s decalcifying Fluid is that by volume part is formulated: 10% 4 parts, nitric acid, 0.5% 3 parts of chromic acid, 3 parts of absolute ethyl alcohols by following reagent.
Described paraffin refers to that fusing point is the flakey paraffin wax of 56 ~ 58 ℃.
In the inventive method, fish gill tissue, through hematoxylin eosin staining, finally uses canada balsam mounting, and prepared histotomy is examined under a microscope, and then records, takes pictures.The morphosis of Dui Yu gill tissue and cell is observed, to form clear, clear fish gill tissue slice map.
Compared with prior art, the invention has the advantages that:
(1) greatly improving Liao Yu gill tissue paraffin prepares fixing effect the present invention and adopts Bouin ' s immobile liquid room temperature to fix, strengthened the penetrating power to tissue, make each component of cell be precipitated fully and solidify, avoid due to destructions such as the material swelling that immersion occurs for a long time, hardening, thereby the morphosis in the time of intactly preserving cells survival, obtains dyeing and observing effect preferably.
(2) the section problem the present invention while greatly improving Liao Yu gill tissue paraffin section and prepare adopts Perenyi ' s liquid as decalcifying Fluid, the fish gill is owing to containing bony structures, if do not carry out decalcification processing, can be harder during section, bad section, the section of preparing with the present invention, not only cuts into slices respond well, and nucleus and cytoplasm fine structure painted all good.
(3) greatly improved and organized effect the present invention of waxdip in soak process, to add the process of dimethylbenzene and paraffin intermixture, accelerated the time that paraffin penetrates into tissue, can make paraffin fully permeate tissue, embedding is respond well.
The present invention is compared with prior paraffin microsection manufacture method, improved conventional dehydration, transparent, the operating process of waxdip, greatly improve the text structure of Liao Yu gill tissue section, solve the problem that Liao Yu gill tissue exists in manufacturing process, be beneficial to the antigen location of immunocytochemical stain, thereby carry out in situ hybridization in the section of Yu gill tissue, the experiment such as immunohistochemistry and immunofluorescence can show Tissue distribution and the celluar localization of some gene and albumen, for further from cell, on gene and protein level, carry out the correlative study of the fish gill practicable condition is provided.
Below in conjunction with embodiment, the preparation method of fish of the present invention gill tissue paraffin section is described further.
embodiment 1
Experiment purpose: by preparation fish gill tissue paraffin section, observe variable concentrations fluoride and expose the pathology of carp gill tissue after 90 d and change.Concrete steps are as follows:
(1) prepare or choose use reagent
It is formulated by volume by following reagent that A prepares Bouin ' s immobile liquid: 15 parts of saturated picric acid, 5 parts of formalin, 1 part, glacial acetic acid.
B prepares Perenyi ' s decalcifying Fluid, and by following reagent, by volume part is formulated: 10% 4 parts, nitric acid, 3 parts of 0.5% chromic acid, 3 parts of absolute ethyl alcohols.
It is the flakey paraffin wax of 56 ~ 58 ℃ that C chooses fusing point.
(2) sampling exposes the anesthesia of carp ice bath after 90 d by variable concentrations fluoride, second gill in clip left side then, and volume is 3 mm * 2, mm * 3 mm, each group is got 2 samples, then puts into the 2ml EP pipe that Bouin ' s immobile liquid is housed.
(3) fixedly guarantee that carp gill tissue material is immersed in after Bouin ' s immobile liquid completely, it at room temperature fixed to 24 h, during change liquid 2 ~ 3 times.
(4) after decalcification organization material is fixed, the material flowing water fixing is rinsed after 24 h, put into Perenyi ' s decalcifying Fluid, under room temperature condition, 5 d are processed in decalcification.
(5) after dehydration decalcification finishes, material is put into ascending gradient ethanol dehydration, successively respectively: 60% alcohol immersion 0.5 h, 70% alcohol immersion 0.5 h, 80% alcohol immersion 0.5 h, 90% alcohol immersion 1 h, 95% alcohol immersion 1 h, 95% alcohol immersion 2 h, soaked in absolute ethyl alcohol 40 min, soaked in absolute ethyl alcohol 40 min.
(6) the transparent material that dehydration is finished by be respectively for twice the dimethylbenzene of 15 min soak carry out transparent.
(7) first paraffin infiltration is immersed the material after transparent in the solution that dimethylbenzene and paraffin are 1:1 by volume and is soaked 30 min, then transfers in the paraffin of 56 ℃ and permeates 4 h.
(8) embedding is converted into small paper box with kraft, and the paraffin first dissolving heating is poured in carton, then rapidly paraffin is permeated good material tangent plane down, the gill lamella and tangent plane laid parallel in carton, cooling forming.
(9) section, by embedded, cooled paraffin mass through over-segmentation, finishing and set, is cut into continuous wax band with microtome by paraffin mass, and wax belt shape is rectangle, and upper and lower two long limits are parallel, and slice thickness is 5 ~ 6 μ m.
(10) exhibition sheet and glutinous sheet cut apart by the wax band cutting the fragment that is suitable, be placed on the distillation water surface of 50 ~ 55 ℃, when section is fully expanded, with the microslide that scribbles glutinous sheet liquid, section is fished for rapidly, section is placed in to the optimal viewing position of microslide, then microslide is placed on to roasting sheet 12 h of constant temperature oven of 40 ℃, the labelled or mark of section after drying, is put in slide storage case.
(11) dewaxing is immersed in dimethylbenzene twice successively by the section of drying, and is 5 min at every turn and carries out dewaxing treatment.
(12) rehydration adopts descending gradient ethanol to carry out rehydration the section after dewaxing, is immersed in successively absolute ethyl alcohol 5 min, absolute ethyl alcohol 5 min, 95% ethanol 3 min, 70% ethanol 5 min, then with clear water, rinses 5 min, distilled water flushing 1 min.
(13) dyeing is put into haematine 15 min that dye by the section after rehydration, then rinses after 1s with tap water, puts into percent by volume and be 1% the ethanol solution hydrochloride differentiation 1s that fades.
(14) redye and will put into after tap water flowing water rinsing 10 min, distilled water flushing 1s through the section of preliminary dyeing, then redye 1 min with Yihong ethanolic solution, then use distilled water rinsing 1-5 s; Finally by crossing 75% ethanol, swing and wash 1-5 s, 95% alcohol flushing 1 min, absolute ethyl alcohol and rinse 1 min, absolute ethyl alcohol and rinse that 1 min, dimethylbenzene rinse 3 min, dimethylbenzene rinses 3 min, complete and redye.
(15) sealing, by the Canadian resin that drips 2-3 in the section of redying and drip, carefully covers cover glass rapidly, can not produce bubble, if there is bubble to squeeze out, then by mounting at room temperature the natural drying carp gill tissue paraffin that is prepare slide.
Yong Gai carp gill tissue slide can be examined under a microscope and the pathology of cinephotomicrography carp gill tissue change.
Claims (4)
- The method for making of 1.Yu gill tissue paraffin section, comprises that step is as follows:(1) fritter that fixedly Jiang Yu gill tissue is cut into 3 mm * 2, mm * 3 mm is soaked in Bouin ' s solution and is fixed, and fixes 24 h under room temperature condition, during change Bouin ' s solution 2 ~ 3 times;(2) the material flowing water that (1) is processed in decalcification rinses 24 h, then immerses in Perenyi ' s decalcifying Fluid, keeps 5 d under room temperature condition;(3) dehydration is immersed ascending gradient ethanol dehydration successively by the material after (2) processing, step is successively: 60% alcohol immersion 0.5 h, 70% alcohol immersion 0.5 h, 80% alcohol immersion 0.5 h, 90% alcohol immersion 1 h, 95% alcohol immersion 1 h, 95% alcohol immersion 2 h, soaked in absolute ethyl alcohol 40 min, soaked in absolute ethyl alcohol 40 min;(4) transparently (3) are handled well to material immerse successively that dimethylbenzene soaks 15 min, dimethylbenzene soaks 15 min;(5) paraffin infiltration is immersed dimethylbenzene and paraffin and in the solution of 1:1, is soaked 30 min by volume handle material well through (4), then transfers in the paraffin of 56 ℃ and permeates 4 h;(6) embedding is converted into small paper box by kraft, and the paraffin first dissolving heating is poured in carton, then rapidly will through (5) handle well material tangent plane down, the gill lamella and tangent plane laid parallel in carton, cooling forming;(7) section, by (6) embedded paraffin mass through over-segmentation, finishing and set, is then cut into continuous wax band through microtome by paraffin mass, and wax belt shape is rectangle, and upper and lower two long limits are parallel, and slice thickness is 5 ~ 6 μ m;(8) the wax band that exhibition sheet and glutinous sheet cut (7) is cut apart the fragment that is suitable, be placed on the distillation water surface of 50 ~ 55 ℃, while observing the abundant expansion of section, with the microslide that scribbles glutinous sheet liquid, section is fished for rapidly, section is controlled to the optimal viewing position of microslide, slide is placed on to roasting sheet 12 h of constant temperature oven of 40 ℃, the labelled or mark of section after drying, is put in slide storage case;(9) dewaxing rehydration is cut into slices and is dewaxed after (8) are processed, be immersed in successively twice of dimethylbenzene, be 5 min at every turn, then carry out descending gradient ethanol rehydration, be immersed in successively absolute ethyl alcohol 5 min, absolute ethyl alcohol 5 min, 95% ethanol 3 min, 70% ethanol 5 min, then with clear water, rinse 5 min, distilled water flushing 1 min;(10) dyeing is put into haematine 15 min that dye by the section of processing through (8), with tap water, rinses after 1s, puts into percent by volume and is 1% ethanol solution hydrochloride and break up the 1s that fades;(11) redye the section that (9) are processed and put into after tap water flowing water rinsing 10 min, distilled water flushing 1s, through Yihong ethanolic solution, redye 1 min, then distilled water rinsing 1-5 s; Finally by crossing 75% ethanol, swing and wash 1-5 s, 95% alcohol flushing 1 min, absolute ethyl alcohol and rinse 1 min, absolute ethyl alcohol and rinse that 1 min, dimethylbenzene rinse 3 min, dimethylbenzene rinses 3 min;(12) be locked in after the Canadian resin that in the section of processing through (10), a 2-3 drips, rapidly cover glass carefully covered, can not produce bubble, if there is bubble to squeeze out, then that mounting is at room temperature natural drying.
- 2. the method for making of fish according to claim 1 gill tissue paraffin section, is characterized in that, described Bouin ' s immobile liquid is formulated by volume by following reagent: 15 parts of saturated picric acid, 5 parts of formalin, 1 part, glacial acetic acid.
- 3. the method for making of fish according to claim 1 gill tissue paraffin section, is characterized in that, described Perenyi ' s decalcifying Fluid is by volume part formulated by following reagent: 10% 4 parts, nitric acid, 0.5% 3 parts of chromic acid, 3 parts of absolute ethyl alcohols.
- 4. the method for making of fish according to claim 1 gill tissue paraffin section, is characterized in that, described paraffin refers to that fusing point is the flakey paraffin wax of 56 ~ 58 ℃.
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