CN107917832A - The production method of fish ovary tissue paraffin section - Google Patents

Abstract

The invention discloses a method for making paraffin slices of fish ovary tissue, which includes material sampling, tissue fixation, dehydration and transparency, wax soaking, embedding, slicing, patching and baking, dewaxing, rehydration, staining cell nuclei, Cytoplasm staining, dehydration, clearing and mounting steps. Compared with the existing method for making paraffin slices, the present invention greatly improves the effect of fish ovary tissue paraffin preparation and fixation, improves the dyeing effect of fish ovary tissue paraffin tissue slices, improves the effects of dehydration and transparency, and greatly improves the performance of fish under the microscope. The integrity and clarity of the ovarian tissue structure solves several problems existing in the prior art in the production process of fish ovarian tissue paraffin sections. It provides supporting conditions for further research on fish ovaries at the gene and protein levels by using research in the fields of morphological pathology, immunology, cell in situ nucleic acid molecular hybridization, and morphometrics.

Description

Preparation method of paraffin section of fish ovary tissue

technical field

The invention belongs to the technical field of making paraffin slices of biological tissues, and in particular relates to a method for making paraffin slices of fish ovarian tissues.

Background technique

Due to its simple operation and low price, paraffin section has always been the most widely used method for making sections in histological microscopic observation. As a traditional biological research technique, paraffin section can not only observe the morphology of normal tissue cells, but also has a wide range of applications in the fields of pathology, immunology, in situ nucleic acid molecular hybridization, and morphometrics. Paraffin sections should go through the steps of fixation, dehydration, transparency, paraffinization, embedding, sectioning, patching, dewaxing, staining, transparency, and mounting to keep the original structure of tissue cells and allow them to be clearly observed and analyzed. However, fish ovaries are rich in yolk, which makes it difficult to make slices. According to the general method in the past, the slices are fragile, and cavities are easily formed in the middle of the slices, so that the structure cannot be seen clearly by microscopic observation. Other immunological and molecular biological analyzes cannot conduct. Therefore, how to improve the existing method for making paraffin slices and solve the problems existing in the process of making fish ovarian tissue slices is a problem to be solved urgently in this field.

Contents of the invention

The purpose of the present invention is to provide a kind of preparation method that can improve and improve the quality of paraffin section of fish ovary in view of some defects that exist in the prior art, this preparation method can make fish ovary tissue and cell structure clear and complete, for further Immunological and molecular biological and morphometric analyzes provide high-quality tissue sections.

The preparation method of fish ovary tissue paraffin section of the present invention, the steps are as follows:

(1) Material sampling The volume of fish ovary tissue sampling material is: 3.6-4 mm×3-3.3mm×3-3.3mm;

(2) Tissue fixation After the fish ovary tissue was fixed with Zamboni’s fixative solution for 8 hours, it was washed with running water for 6 hours to wash off the fixative solution;

(3) Dehydration and transparency Treat the tissues treated in (2) with the following reagents in turn: 50% ethanol for 10 minutes, 70% ethanol for 15 minutes, 80% ethanol for 15 minutes, 95% ethanol for 90 minutes, 95% ethanol for 90 minutes, 2 parts of absolute ethanol + 1 part of n-butanol for 60 minutes, 1 part of absolute ethanol + 1 part of n-butanol for 60 minutes, n-butanol for 45 minutes, and n-butanol for 45 minutes;

(4) Wax immersion Put the tissue sample treated in (3) into a paraffin wax immersion box for immersion wax, and treat it with the following reagents in turn: 1 part of n-butanol +: 1 part of paraffin for 30 minutes, paraffin for 90 minutes, Paraffin 90min

(5) Paraffin embedding: Put the tissue sample treated in (4) into an embedding box with melted paraffin for embedding. After the paraffin is naturally cooled and solidified, take out the embedded wax block from the box for trimming;

(6) Slicing: Glue the trimmed wax block (5) to the prepared small wooden block, then fix it on the slicer, adjust it parallel to the slicer knife, and cut into wax strips;

(7) Sticker and baked slices. Among the slices prepared in (6), select the wax tape with complete tissue samples. The smooth side of the wax tape is facing down. Remove the acid-treated glass slides from the water, arrange the tissue samples neatly on the glass slides, mark the slices, place them on the slice rack, bake the slices in an oven at 45°C for 5.5-6 hours, and cool naturally;

(8) Dewaxing Put the slide holder into the staining jar, and treat it with the following reagents in turn: xylene for 10 minutes, xylene for 10 minutes;

(9) Rehydration. The slices treated in (8) were treated with the following reagents in sequence: absolute ethanol for 2 minutes, absolute ethanol for 2 minutes, 95% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, tap water to slowly rinse, distilled water for 2 minutes ;

(10) Stain the cell nuclei. Pour the prepared Harris' hematoxylin solution into the staining jar, put the slices treated in (9) into the staining jar at room temperature for 10-20 minutes, and then treat with the following reagents in sequence: slowly wash away the floating color with tap water for 1 minute , 1% hydrochloric acid alcohol differentiation 10s, 1% ammonia water 10s, distilled water 1~2min;

(11) Stain the cytoplasm. Treat the sections of the cell nuclei stained in (10) with the following reagents in sequence: eosin staining solution for 5 minutes, slowly rinse with tap water for 30 seconds;

(12) Dehydration: Treat the sections after (11) with the following reagents in turn: 70% ethanol for 30s, 80% ethanol for 30s, 95% ethanol for 2 minutes, absolute ethanol for 2 minutes, absolute ethanol for 2 minutes;

(13) Transparency Treat the slices through (12) with the following reagents in turn: xylene for 5 minutes, xylene for 5 minutes;

(14) Mounting After drying the transparent slices in (13), drop neutral gum, and slowly lower the cover slip from one side. Once dry, scrape off excess gum with a razor blade.

Described Zamboni's fixing solution is made up of following reagent, paraformaldehyde 25g, saturated picric acid 200ml, Karasson-Schwlt's phosphate buffered saline to 1000ml.

The paraffin refers to the scale-like section paraffin with a melting point of 52-56°C.

The Harris' hematoxylin solution is made up of the following reagents: 2 g of hematoxylin, 30 ml of absolute ethanol, 40 g of potassium aluminum sulfate, 500 ml of distilled water, 1.5 g of red mercuric oxide, and 15 ml of glacial acetic acid.

In the method of the present invention, the fish ovary tissue is stained with hematoxylin-eosin, and finally sealed with neutral gum, and the prepared tissue section is observed under a microscope and photographed. Observe the morphological structure of fish ovary tissue and cells to form a clear and complete fish ovary tissue slice map.

Compared with the prior art, the present invention has the following advantages: (1) The present invention uses Zamboni's (Stefanini's) fixative solution to fix at room temperature, which increases its penetrating power and fixation effect on tissues, so that tissues can be fixed quickly and avoids fixation of tissue samples If it hardens for too long, the slices will be fragile, and the fixative can preserve more tissue antigens, providing a basis for further immunocytochemistry and molecular biology analysis, and greatly improving the effect of paraffin preparation and fixation of fish ovary tissues . (2) The present invention adopts Harris' hematoxylin solution as the staining agent of cell nuclei, which can make the fine structure of cell nuclei stain bright and uniform, and greatly improve the staining effect of paraffin tissue sections of fish ovary tissue. (3) The present invention uses a mixture of absolute ethanol and n-butanol. The gentle dehydration ability of n-butanol can effectively improve the shrinkage and hardening of tissues caused by excessive dehydration of absolute ethanol alone and affect the section observation. Greatly improved the effect of dehydration. (4) The present invention uses n-butanol instead of traditional xylene as a transparent agent. The mild transparent ability of n-butanol avoids the strong transparent ability of xylene and the short time that makes the tissue shrink and become brittle, which leads to the fragmentation of tissue slices. situation, greatly improving the effect of transparency. (5) Compared with the traditional paraffin section production method, the present invention improves the conventional dehydration, transparency, and wax-immersion operation process, greatly improves the structural clarity of the fish ovary tissue section, and solves the problems existing in the production process of the fish ovary tissue. Moreover, the application research in the fields of morphological pathology, immunology, cell in situ nucleic acid molecular hybridization, and morphometry provides supporting conditions for further research on fish ovaries at the gene and protein levels.

The preparation method of the fish ovary tissue paraffin section of the present invention will be further described in conjunction with the examples below.

Example 1

The invention is used to make paraffin sections of fish ovary tissue, and to observe the pathological changes and immunohistochemical localization of carp ovary tissue after exposure to different concentrations of copper for 30 days. Specific steps are as follows:

1. Preparation of related reagents

(1) Preparation of Zamboni’s fixative solution Zamboni’s fixative solution is made up of the following reagents, paraformaldehyde 25g, saturated picric acid 200ml, Karasson-Schwlt’s phosphate buffer to 1000ml.

(2) Prepare paraffin. Purchase paraffin wax with a melting point of 52-56°C in advance.

(3) Preparation of Harris' hematoxylin solution Harris' hematoxylin solution is prepared from the following reagents: 2g of hematoxylin, 30ml of absolute ethanol, 40g of potassium aluminum sulfate, 500ml of distilled water, 1.5g of red mercuric oxide, and 15ml of glacial acetic acid.

2. Paraffin section making

(1) Material sampling The volume of fish ovary tissue sampling material is: 3.8-4 mm×3-3.2mm×3-3.2mm;

(2) Tissue fixation After the fish ovary tissue was fixed with Zamboni’s fixative solution for 8 hours, it was washed with running water for 6 hours to wash off the fixative solution;

(3) Dehydration and transparency Treat the tissues treated in (2) with the following reagents in turn: 50% ethanol for 10 minutes, 70% ethanol for 15 minutes, 80% ethanol for 15 minutes, 95% ethanol for 90 minutes, 95% ethanol for 90 minutes, 2 parts of absolute ethanol + 1 part of n-butanol for 60 minutes, 1 part of absolute ethanol + 1 part of n-butanol for 60 minutes, n-butanol for 45 minutes, and n-butanol for 45 minutes;

(4) Wax immersion Put the tissue sample treated in (3) into a paraffin wax immersion box for immersion wax, and treat it with the following reagents in turn: 1 part of n-butanol +: 1 part of paraffin for 30 minutes, paraffin for 90 minutes, Paraffin 90min

(5) Paraffin embedding: Put the tissue sample treated in (4) into an embedding box with melted paraffin for embedding. After the paraffin is naturally cooled and solidified, take out the embedded wax block from the box for trimming;

(6) Slicing: Glue the trimmed wax block (5) to the prepared small wooden block, then fix it on the slicer, adjust it parallel to the slicer knife, and cut into wax strips;

(7) Sticker and baked slices. Among the slices prepared in (6), select the wax tape with complete tissue samples. The smooth side of the wax tape is facing down. Remove the acid-treated glass slides from the water, arrange the tissue samples neatly on the glass slides, mark the slices, place them on the slice rack, bake the slices in an oven at 45°C for 5.5-6 hours, and cool naturally;

(8) Dewaxing Put the slide holder into the staining jar, and treat it with the following reagents in turn: xylene for 10 minutes, xylene for 10 minutes;

(9) Rehydration. The slices treated in (8) were treated with the following reagents in sequence: absolute ethanol for 2 minutes, absolute ethanol for 2 minutes, 95% ethanol for 3 minutes, 80% ethanol for 3 minutes, 70% ethanol for 3 minutes, tap water to slowly rinse, distilled water for 2 minutes ;

(10) Stain the cell nuclei. Pour the prepared Harris' hematoxylin solution into the staining jar, put the slices treated in (9) into the staining jar at room temperature for 10-20 minutes, and then treat with the following reagents in sequence: slowly wash away the floating color with tap water for 1 minute , 1% hydrochloric acid alcohol differentiation 10s, 1% ammonia water 10s, distilled water 1~2min;

(11) Stain the cytoplasm. Treat the sections of the cell nuclei stained in (10) with the following reagents in sequence: eosin staining solution for 5 minutes, slowly rinse with tap water for 30 seconds;

(12) Dehydration: Treat the sections after (11) with the following reagents in turn: 70% ethanol for 30s, 80% ethanol for 30s, 95% ethanol for 2 minutes, absolute ethanol for 2 minutes, absolute ethanol for 2 minutes;

(13) Transparency Treat the slices through (12) with the following reagents in turn: xylene for 5 minutes, xylene for 5 minutes;

(14) Mounting After drying the transparent slices in (13), drop neutral gum, slowly put down the cover glass from one side, after drying, scrape off the excess gum with a blade.

The pathological changes of the carp ovary tissue can be observed, morphometrically analyzed and microphotographed under a microscope by using the sliced carp ovary tissue.

Claims (4)

1. the production method of fish ovary tissue paraffin section, step are as follows：

（1）Material sampling fish ovaries sample of tissue material volume be：3.6-4 mm×3-3.3mm×3-3.3mm；

（2）The fixed fish ovaries tissue of tissue through Zamboni ' s fixers fix 8 it is small when after, flowing water rinse 6 it is small when, it is solid to wash away it Determine liquid；

（3）Dehydration and transparent general（2）The tissue of processing is successively with following agent treatment：50% ethanol 10min, 70% ethanol 15min, 80% ethanol 15min, 95% ethanol 90min, 95% ethanol 90min, 2 parts+n-butanol of absolute ethyl alcohol 1 part of 60 min, it is anhydrous 1 part+n-butanol of ethanol, 1 part of 60 min, n-butanol 45min, n-butanol 45min；

（4）Waxdip will be through（3）Processed tissue sample, which is put into the waxdip box for melted paraffin, carries out waxdip, successively with following Agent treatment：1 part of n-butanol+:1 part of 30min of paraffin, paraffin 90min, paraffin 90min

（5）Paraffin embedding will be through（4）Processed tissue sample, which is put into the embedded box for melted paraffin, to be embedded, and waits paraffin After natural cooling solidification, embedded wax stone is taken out from box finishing；

（6）Section will（5）The wax stone trimmed is cemented on ready wood particle, is then fixed on slicer, with section Knife is tuned into parallel, is cut into wax band；

（7）Patch and roasting piece exist（6）In the section of preparation, selection has the wax band of complete tissue sample, and wax band is smooth downwards, puts Enter after opening up piece in 40 ~ 42 DEG C of water baths, pulled out with the processed glass slide of poly-D-lysine from water, make tissue sample proper alignment On glass slide, mark is performed in section, is placed in slide holding frame, the roasting piece 5.5-6h of 45 DEG C of baking oven, natural cooling；

（8）The slide holding frame for being placed with glass slide is put into staining jar by dewaxing, successively with following agent treatment：Dimethylbenzene 10min, Dimethylbenzene 10min；

（9）Rehydration will be through（8）The section of processing is successively with following agent treatment：Absolute ethyl alcohol 2min, absolute ethyl alcohol 2min, 95% Ethanol 3min, 80% ethanol 3min, 70% ethanol 3min, tap water slowly rinse, distilled water 2min；

（10）The HarrisShi haematoxylin liquid configured is poured into staining jar by dye nucleus, at room temperature warp（9）The section of processing 10 ~ 20min of staining jar is put into, then successively with following agent treatment：Tap water slowly washes away loose colour 1min, 1% hydrochloride alcohol point Change 10s, 1% ammonium hydroxide, 10 s, 1 ~ 2min of distilled water；

（11）Contaminating cytoplasm will be through（10）The section of nucleus has been contaminated successively with following agent treatment：Eosin stain 5min, originally Water slowly rinses loose colour 30s；

（12）Dehydration will be through（11）Section successively with following agent treatment：70% ethanol 30s, 80% ethanol 30s, 95% ethanol 2min, absolute ethyl alcohol 2min, absolute ethyl alcohol 2min；

（13）It is transparent will be through（12）Section successively with following agent treatment：Dimethylbenzene 5min, dimethylbenzene 5min；

（14）Sealing will be through（13）After transparent good section is dried, neutral gum is dripped, coverslip is slowly put down from side, dries Afterwards, unnecessary natural gum is scraped off with blade.

2. the production method of fish ovary tissue paraffin section according to claim 1, it is characterised in that described Zamboni ' s fixers are made into by following reagent, paraformaldehyde 25g, saturation picric acid 200ml, Karasson-Schwlt ' s Phosphate buffer is to 1000ml.

3. the production method of fish ovary tissue paraffin section according to claim 1, it is characterised in that the paraffin Refer to the flakey paraffin wax that fusing point is 52 ~ 56 DEG C.

4. the production method of fish ovary tissue paraffin section according to claim 1, it is characterised in that described HarrisShi haematoxylins liquid is made into by following reagent, haematoxylin 2g, absolute ethyl alcohol 30ml, alum 40g, distilled water 500ml, Mercury oxide red 1.5g, glacial acetic acid 15ml.