CN112129600A - Method for preparing frozen section of horse, donkey and mule testis - Google Patents
- ️Fri Dec 25 2020
技术领域technical field
本发明涉及病理切片制备技术领域，具体涉及一种用于制备马驴骡睾丸冰冻切片的方法。The invention relates to the technical field of preparation of pathological slices, in particular to a method for preparing frozen slices of testicles of horses, donkeys and mules.
背景技术Background technique
冰冻切片是临床病理检查中常用的快速制片方法，其是在低温条件下使组织快速冷冻到一定的程度后，进行制作切片的一种技术，由于冰冻切片制作过程简单、快建、制作时间短，在抗原的保存上也有显著优势，在一些实验室也会常被用来开展组织水平的生理、病理和分子学研究。Frozen section is a commonly used rapid preparation method in clinical pathological examination. It is a technique for making sections after the tissue is rapidly frozen to a certain extent under low temperature conditions. It also has significant advantages in the preservation of antigens, and is often used in some laboratories to carry out physiological, pathological and molecular studies at the tissue level.
目前，冰冻切片制作过程中，需要对样品的操作与把控都必须很小心，如果出现刀痕、冰晶、切片不完整、厚度不适宜、固定不好等情况，常会导致细胞肿胀变形，影响观察切片状态。在科研实验的免疫组化学染色中，冰冻切片能较好地保存细胞抗原活性，但是因为大动物睾丸体积普遍比小鼠等模式动物睾丸大，组织水分含量多，制作冰冻切片时容易冰晶而影响切片结果；在利用小鼠、大鼠等模式动物器官组织制作冰冻切片方案，进行制作马睾丸冰冻切片时也不易成功。传统液氮法制作切片，因为马睾丸体积大水分含量高，取样速冻时容易冰晶，同时容易产生体积的骤变而引起整体变形会引起睾丸组织细胞变形，各类细胞无法辨认，无法观察睾丸形态和细胞核。At present, during the production of frozen sections, it is necessary to be very careful in the operation and control of the samples. If knife marks, ice crystals, incomplete sections, unsuitable thickness, or poor fixation occur, cells will often swell and deform, which will affect the observation. slice state. In the immunohistochemical staining of scientific research experiments, frozen sections can better preserve the activity of cell antigens, but because the testis of large animals is generally larger than the testis of model animals such as mice, and the tissue moisture content is high, it is easy to be affected by ice crystals when making frozen sections. Sectioning results; it is not easy to succeed when using the mouse, rat and other model animal organs and tissues to make frozen section plans, and to make frozen sections of horse testis. The traditional liquid nitrogen method for making slices, because the horse testis is large in size and high in water content, it is easy to ice crystals when sampling and quick-freezing, and it is easy to produce a sudden change in volume and cause overall deformation, which will cause the deformation of testicular tissue cells, and various types of cells cannot be identified. and nucleus.
综上所述，在对马睾丸进行组织免疫荧光技术的时，亟需研发出一种新的对该组织冰冻切片的制作方法。In conclusion, when performing tissue immunofluorescence on horse testis, it is urgent to develop a new method for making frozen sections of the tissue.
发明内容SUMMARY OF THE INVENTION
为此，本发明提供一种用于制备马驴骡睾丸冰冻切片的方法。To this end, the present invention provides a method for preparing frozen sections of testis of donkey and mule.
为了实现上述目的，本发明提供如下技术方案：In order to achieve the above object, the present invention provides the following technical solutions:
一种用于制备马驴骡睾丸冰冻切片的方法，所述方法包括将块状睾丸组织置于Bouin固定液固定后，将固定后的睾丸组织在蔗糖溶液中脱水，将脱水后的睾丸组织包埋，冷冻切片，得到睾丸冷冻切片。A method for preparing frozen sections of testis of horse, donkey and mule, the method comprising placing block testis tissue in Bouin fixative for fixation, dehydrating the fixed testis tissue in sucrose solution, and wrapping the dehydrated testis tissue in a sucrose solution. Buried and cryosectioned to obtain testicular cryosections.
本发明的一个实施例中，所述脱水过程为：将固定后的睾丸组织依次在质量百分数为5％蔗糖、10％蔗糖、20％蔗糖分别处理30min；In an embodiment of the present invention, the dehydration process is as follows: the fixed testis tissue is sequentially treated with mass percentages of 5% sucrose, 10% sucrose, and 20% sucrose for 30 minutes;
再依次在第一混合液、第二混合液以及第三混合液中，分别处理30min；Then in the first mixed solution, the second mixed solution and the third mixed solution, respectively, for 30min;
最后在100％OCT中过夜；Finally overnight in 100% OCT;
其中，第一混合液为20％蔗糖与OCT体积比为2:1混合而成；Wherein, the first mixed solution is formed by mixing 20% sucrose and OCT in a volume ratio of 2:1;
第二混合液为20％蔗糖与OCT体积比为1:1混合而成；The second mixed solution is made by mixing 20% sucrose and OCT with a volume ratio of 1:1;
第三混合液为20％蔗糖与OCT体积比为1:2混合而成。The third mixed solution is formed by mixing 20% sucrose and OCT in a volume ratio of 1:2.
本发明的一个实施例中，所述包埋的过程为：In one embodiment of the present invention, the process of the embedding is:
向经过透明处理后的睾丸组织中加入OCT包埋剂冷冻20-25min或者液氮速冻20-25min。Add OCT embedding medium to the clarified testis tissue for 20-25min or liquid nitrogen quick-freeze for 20-25min.
本发明的一个实施例中，所述块状睾丸组织的大小为1-2cm3。In an embodiment of the present invention, the size of the bulk testicular tissue is 1-2 cm 3 .
本发明的一个实施例中，所述块状睾丸组织的固定时间为24h。In an embodiment of the present invention, the fixation time of the bulk testicular tissue is 24 hours.
本发明的一个实施例中，所述睾丸冷冻切片的切片厚度为4.5μm。In one embodiment of the present invention, the section thickness of the testis frozen section is 4.5 μm.
本发明具有如下优点：The present invention has the following advantages:
本发明的组织固定法可以使蛋白变性、凝固、沉淀、保持细胞、组织原有的形态结构防止组织的自溶与腐败，细胞内的酶类转变为不溶性物质，以保持原有的结构，组织固定后，均呈一定的硬化状态，增加了组织的韧性，而且不易变形，保持了细胞完整，在HE染色和DAPI核染后均能呈现出良好的细胞组织形态结构。The tissue fixation method of the present invention can denature, coagulate, and precipitate proteins, maintain the original morphological structure of cells and tissues, prevent autolysis and corruption of tissues, and convert enzymes in cells into insoluble substances to maintain the original structure and tissue. After fixation, they all showed a certain hardening state, which increased the toughness of the tissue, and was not easy to deform.
附图说明Description of drawings
为了更清楚地说明本发明的实施方式或现有技术中的技术方案，下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地，下面描述中的附图仅仅是示例性的，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图引伸获得其它的实施附图。In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, other implementation drawings can also be obtained according to the extension of the drawings provided without creative efforts.
图1为本发明提供的传统方法制备的冷冻切片和本发明方法制备的冷冻切片HE染色显微镜照片图，其中，A为传统方法制备的冷冻切片HE染色，B为本发明方法制备的冷冻切片HE染色；Fig. 1 is the frozen section prepared by the traditional method provided by the present invention and the frozen section prepared by the inventive method HE staining micrograph, wherein, A is the frozen section HE staining prepared by the traditional method, B is the frozen section prepared by the inventive method HE staining dyeing;
图2为本发明提供的传统方法制备的冷冻切片和本发明方法制备的冷冻切片DAPI染色显微镜照片图；其中，A为传统方法制备的冷冻切片DAPI染色，B为本发明方法制备的冷冻切片DAPI染色。Fig. 2 is the frozen section prepared by the traditional method provided by the present invention and the frozen section DAPI dyeing micrograph prepared by the method of the present invention; wherein, A is the frozen section DAPI dyeing prepared by the traditional method, and B is the frozen section DAPI prepared by the method of the present invention. dyeing.
具体实施方式Detailed ways
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The embodiments of the present invention are described below by specific specific embodiments. Those who are familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
本发明中，OCT：optimal cutting temperature compound，是一种聚乙二醇和聚乙烯醇的水溶性混合物，用于免疫组化实验室中，其用途是在冰冻切片时支撑组织，以增加组织的连续性，减少皱折及碎裂。In the present invention, OCT: optimal cutting temperature compound, is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol, used in immunohistochemical laboratories, and its purpose is to support tissue during frozen sectioning to increase the continuity of tissue resistance, reduce wrinkling and chipping.
本发明中，Bouin固定液是由苦味酸饱和液(1.22％)、甲醛和冰醋酸按照15:5:1的比例混合而成。In the present invention, the Bouin fixative is prepared by mixing picric acid saturated solution (1.22%), formaldehyde and glacial acetic acid in a ratio of 15:5:1.
本发明实施例中，所采用的实验仪器：烘箱(上海一恒)、水浴锅、通风柜、体视显微镜、冻切片机(Leica CM1950)、摇床、病理及粘附载玻片(Medvid PC2-301)、盖玻片、Nikon正置显微镜、倒置显微镜(Zeiss Axio Observer D1)。In the embodiment of the present invention, the experimental instruments used: oven (Shanghai Yiheng), water bath, fume hood, stereo microscope, cryostat (Leica CM1950), shaking table, pathological and adhesive slides (Medvid PC2 -301), coverslip, Nikon upright microscope, inverted microscope (Zeiss Axio Observer D1).
实施例1Example 1
本实施例提供的用于制备马驴骡睾丸冰冻切片的方法，其包括以下步骤：The method for preparing the frozen section of the mule testis provided by the present embodiment comprises the following steps:
步骤一、马睾丸组织的固定Step 1. Fixation of equine testis tissue
将新鲜四岁马睾丸组织快速切成1cm3后，放入Bouin固定液，固定时间为24h，得到固定后的马睾丸组织。The fresh four-year-old horse testis tissue was quickly cut into 1cm 3 and then put into Bouin's fixative for 24h to obtain the fixed horse testis tissue.
步骤二、对固定后的马睾丸组织脱水Step 2. Dehydration of the fixed horse testis tissue
将固定后的马睾丸组织在质量百分数为5％蔗糖处理30min，10％蔗糖处理30min，20％蔗糖处理30min；再依次将马睾丸组织在第一混合液处理30min，第二混合液处理30min以及第三混合液中处理30min；最后将睾丸组织在100％OCT中过夜，得到脱水后的睾丸组织。The fixed horse testis tissue was treated with 5% sucrose for 30 min, 10% sucrose for 30 min, and 20% sucrose for 30 min; then the horse testis tissue was treated with the first mixed solution for 30 min, the second mixed solution for 30 min and The third mixed solution was treated for 30 min; finally, the testis tissue was placed in 100% OCT overnight to obtain dehydrated testis tissue.
其中，第一混合液为20％蔗糖与OCT体积比为2:1混合而成；第二混合液为20％蔗糖与OCT体积比为1:1混合而成；第三混合液为20％蔗糖与OCT体积比为1:2混合而成。Among them, the first mixed solution is made by mixing 20% sucrose and OCT with a volume ratio of 2:1; the second mixed solution is made by mixing 20% sucrose and OCT with a volume ratio of 1:1; the third mixed solution is 20% sucrose It is mixed with OCT in a volume ratio of 1:2.
步骤三、脱水后的马睾丸组织的包埋Step 3. Embedding of dehydrated horse testis tissue
将冰冻切片机温度提前设置为-25℃，在放置脱水后的马睾丸组织前，先在托盘上加入少量OCT包埋剂，待其微冻时，再将在100％OCT里的马睾丸组织放在托盘上，同时加入OCT包埋剂，使马睾丸组织完全埋在OCT包埋剂中，立即放入预先调好温度的恒冷箱切片机箱内，冷冻20min或者用液氮速冻，即可切片。Set the temperature of the cryostat to -25°C in advance. Before placing the dehydrated horse testis tissue, add a small amount of OCT embedding medium on the tray. When it is slightly frozen, the horse testis tissue in 100% OCT is placed. Put it on the tray, and add OCT embedding medium at the same time, so that the horse testis tissue is completely embedded in the OCT embedding medium, immediately put it into the pre-adjusted temperature cryostat section box, freeze for 20 minutes or use liquid nitrogen to quickly freeze. slice.
步骤四、对包埋的睾丸组织进行切片Step 4. Section the embedded testis tissue
首先，对锋利的切片刀调节出合适的角度安装在刀架上，之后将已经包埋好的组织托盘安装在冷冻切片仪上进行切片，马睾丸组织冷冻切片厚度为4.5μm，之后将马睾丸组织切片在室温晾干5min，得到马睾丸组织冷冻切片。First, adjust the sharp slicing knife to a suitable angle and install it on the knife holder, then install the embedded tissue tray on the cryostat for slicing. The thickness of the frozen section of the horse testis tissue is 4.5 μm, and then the horse testis is sliced. The tissue sections were air-dried at room temperature for 5 min to obtain frozen sections of horse testis tissue.
实施例2Example 2
本实施例提供的用于制备马驴骡睾丸冰冻切片的方法，其包括以下步骤：The method for preparing the frozen section of the mule testis provided by the present embodiment comprises the following steps:
步骤一、马睾丸组织的固定Step 1. Fixation of equine testis tissue
将新鲜四岁马睾丸组织快速切成1cm3后，放入Bouin固定液，固定时间为24h，得到固定后的马睾丸组织。The fresh four-year-old horse testis tissue was quickly cut into 1cm 3 and then put into Bouin's fixative for 24h to obtain the fixed horse testis tissue.
步骤二、对固定后的马睾丸组织脱水Step 2. Dehydration of the fixed horse testis tissue
将固定后的马睾丸组织在质量百分数为5％蔗糖处理30min，10％蔗糖处理30min，20％蔗糖处理30min；再依次将马睾丸组织在第一混合液处理30min、第二混合液处理30min以及第三混合液中处理30min；最后将睾丸组织在100％OCT中过夜，得到脱水后的睾丸组织。The fixed horse testis tissue was treated with 5% sucrose for 30 min, 10% sucrose for 30 min, and 20% sucrose for 30 min; then the horse testis tissue was treated in the first mixed solution for 30 min, the second mixed solution for 30 min and The third mixed solution was treated for 30 min; finally, the testis tissue was placed in 100% OCT overnight to obtain dehydrated testis tissue.
其中，第一混合液为20％蔗糖与OCT体积比为2:1混合而成；第二混合液为20％蔗糖与OCT体积比为1:1混合而成；第三混合液为20％蔗糖与OCT体积比为1:2混合而成。Among them, the first mixed solution is made by mixing 20% sucrose and OCT with a volume ratio of 2:1; the second mixed solution is made by mixing 20% sucrose and OCT with a volume ratio of 1:1; the third mixed solution is 20% sucrose It is mixed with OCT in a volume ratio of 1:2.
步骤三、脱水后的马睾丸组织的包埋Step 3. Embedding of dehydrated horse testis tissue
将冰冻切片机温度提前设置为-25℃，在放置脱水后的马睾丸组织前，先在托盘上加入少量OCT包埋剂，待其微冻时，再将在100％OCT里的马睾丸组织放在托盘上，同时加入OCT包埋剂，使马睾丸组织完全埋在OCT包埋剂中，立即放入预先调好温度的恒冷箱切片机箱内，冷冻22min或者用液氮速冻，完成对马睾丸组织的包埋。Set the temperature of the cryostat to -25°C in advance. Before placing the dehydrated horse testis tissue, add a small amount of OCT embedding medium on the tray. When it is slightly frozen, the horse testis tissue in 100% OCT is placed. Put it on the tray, and add OCT embedding medium at the same time, so that the horse testis tissue is completely embedded in the OCT embedding medium, immediately put it into the pre-adjusted temperature cryostat section box, freeze for 22 minutes or use liquid nitrogen to quickly freeze, complete the alignment. Embedding of equine testicular tissue.
步骤四、对包埋的睾丸组织进行切片Step 4. Section the embedded testis tissue
首先，对锋利的切片刀调节出合适的角度安装在刀架上，之后将已经包埋好的组织托盘安装在冷冻切片仪上进行切片，马睾丸组织冷冻切片厚度为4.5μm，之后将马睾丸组织切片在室温晾干5min，得到马驴骡睾丸组织冷冻切片。First, adjust the sharp slicing knife to a suitable angle and install it on the knife holder, then install the embedded tissue tray on the cryostat for slicing. The thickness of the frozen section of the horse testis tissue is 4.5 μm, and then the horse testis is sliced. The tissue sections were air-dried at room temperature for 5 min to obtain frozen sections of testis tissue of horse, donkey and mule.
实施例3Example 3
本实施例提供的用于制备马驴骡睾丸冰冻切片的方法，其包括以下步骤：The method for preparing the frozen section of the mule testis provided by the present embodiment comprises the following steps:
步骤一、马睾丸组织的固定Step 1. Fixation of equine testis tissue
将新鲜四岁马睾丸组织快速切成1cm3后，放入Bouin固定液，固定时间为24h，得到固定后的马睾丸组织。The fresh four-year-old horse testis tissue was quickly cut into 1cm 3 and then put into Bouin's fixative for 24h to obtain the fixed horse testis tissue.
步骤二、对固定后的马睾丸组织脱水Step 2. Dehydration of the fixed horse testis tissue
将固定后的马睾丸组织在质量百分数为5％蔗糖处理30min，10％蔗糖处理30min，20％蔗糖处理30min；再依次将处理后的马睾丸组织在第一混合液处理30min、第二混合液处理30min以及第三混合液中处理30min；最后将马睾丸组织在100％OCT中过夜，得到脱水后的睾丸组织。The fixed horse testis tissue was treated with 5% sucrose for 30 min, 10% sucrose for 30 min, and 20% sucrose for 30 min; then the treated horse testis tissue was treated in the first mixed solution for 30 min and the second mixed solution in turn. Treated for 30 min and in the third mixed solution for 30 min; finally, the horse testis tissue was placed in 100% OCT overnight to obtain dehydrated testis tissue.
其中，第一混合液为20％蔗糖与OCT体积比为2:1混合而成；第二混合液为20％蔗糖与OCT体积比为1:1混合而成；第三混合液为20％蔗糖与OCT体积比为1:2混合而成。Among them, the first mixed solution is made by mixing 20% sucrose and OCT with a volume ratio of 2:1; the second mixed solution is made by mixing 20% sucrose and OCT with a volume ratio of 1:1; the third mixed solution is 20% sucrose It is mixed with OCT in a volume ratio of 1:2.
步骤三、脱水后的马睾丸组织的包埋Step 3. Embedding of dehydrated horse testis tissue
将冰冻切片机温度提前设置为-25℃，在放置脱水后的马睾丸组织前，先在托盘上加入少量OCT包埋剂，待其微冻时，再将在100％OCT里的马睾丸组织放在托盘上，同时加入OCT包埋剂，使马睾丸组织完全埋在OCT包埋剂中，立即放入预先调好温度的恒冷箱切片机箱内，冷冻25min或者用液氮速冻，完成对马睾丸组织的包埋。Set the temperature of the cryostat to -25°C in advance. Before placing the dehydrated horse testis tissue, add a small amount of OCT embedding medium on the tray. When it is slightly frozen, the horse testis tissue in 100% OCT is placed. Put it on the tray, and add OCT embedding medium at the same time, so that the horse testis tissue is completely embedded in the OCT embedding medium, immediately put it into the pre-adjusted temperature cryostat section box, freeze for 25 minutes or use liquid nitrogen to quickly freeze, complete the alignment. Embedding of equine testicular tissue.
步骤四、对包埋的马睾丸组织进行切片Step 4. Slice the embedded horse testis tissue
首先，对锋利的切片刀调节出合适的角度安装在刀架上，之后将已经包埋好的组织托盘安装在冷冻切片仪上进行切片，马睾丸组织冷冻切片厚度为4.5μm，之后将马睾丸组织切片在室温晾干5min，得到马驴骡睾丸组织冷冻切片。First, adjust the sharp slicing knife to a suitable angle and install it on the knife holder, then install the embedded tissue tray on the cryostat for slicing. The thickness of the frozen section of the horse testis tissue is 4.5 μm, and then the horse testis is sliced. The tissue sections were air-dried at room temperature for 5 min to obtain frozen sections of testis tissue of horse, donkey and mule.
对比例1、冷冻切片HE染色Comparative Example 1. HE staining of frozen sections
本对比例1与实施例1的区别在于，将马睾丸组织液氮法冰冻后，直接切片，得到冷冻切片，将该冷冻切片直接用于HE染色。The difference between this Comparative Example 1 and Example 1 is that after freezing the horse testis tissue in liquid nitrogen, it was directly sectioned to obtain a frozen section, and the frozen section was directly used for HE staining.
对比例2、冷冻切片DAPI染色Comparative Example 2. DAPI staining of frozen sections
本对比例2与实施例2的区别在于，将马睾丸组织液氮冰冻后，直接切片，得到冷冻切片，并将该冷冻切片直接用于DAPI染色。The difference between this Comparative Example 2 and Example 2 is that after freezing the horse testis tissue in liquid nitrogen, it is directly sectioned to obtain a frozen section, and the frozen section is directly used for DAPI staining.
试验例1、HE染色Test Example 1. HE staining
将实施例1和对比例1制备的冷冻切片使用苏木精染液孵育6min后，用自来水冲洗10min，分化液中浸泡1min，自来水中浸泡15min，孵育伊红染液染3min并再次在自来水中浸泡5min之后，在95％乙醇、无水乙醇和二甲苯中各浸泡两次，每次为1min，最后进行封片，显微镜观察。The frozen sections prepared in Example 1 and Comparative Example 1 were incubated with hematoxylin staining solution for 6 min, rinsed with tap water for 10 min, soaked in differentiation solution for 1 min, soaked in tap water for 15 min, incubated with eosin staining solution for 3 min, and then immersed in tap water again. After soaking for 5 min, soak in 95% ethanol, absolute ethanol and xylene for two times, each for 1 min, and finally mount the slide and observe under microscope.
如图1所示，通过对比，本发明实施例1通过固定法制备的马睾丸冰冻切片是更加适合的方案，对比例1制备的冰冻切片会引起睾丸组织细胞的变形，无法观察睾丸性状和细胞核。本发明实施例1制备的冰冻切片经过HE染色后，其可以有效地呈现马睾丸组织细胞的形态与结构，可以作为更进一步地免疫组化等原位分子技术的前期步骤，用于组织化学水平的生理和病理研究。As shown in Figure 1, by comparison, the frozen section of horse testis prepared by the fixation method in Example 1 of the present invention is a more suitable solution. The frozen section prepared in Comparative Example 1 will cause deformation of testicular tissue cells, and it is impossible to observe testicular traits and nuclei. . After the frozen section prepared in Example 1 of the present invention is stained with HE, it can effectively present the morphology and structure of equine testicular tissue cells, and can be used as a preliminary step for further in situ molecular techniques such as immunohistochemistry. Physiological and pathological research.
试验例2、DAPI染色Test example 2, DAPI staining
将实施例2和对比例2制备的冷冻切片，用PBS洗5min后，孵育Triton-X100 30min，再用PBS洗3×5min，37℃烘箱内用5％BSA封闭2h，再孵育DAPI 30min，PBS洗3×5min，最后用防荧光淬灭剂封片，并用荧光显微镜观片，观看完的切片可以在-20℃冰箱保存。The frozen sections prepared in Example 2 and Comparative Example 2 were washed with PBS for 5 min, incubated with Triton-X100 for 30 min, washed with PBS for 3 × 5 min, blocked with 5% BSA in a 37°C oven for 2 h, incubated with DAPI for 30 min, and then incubated with PBS for 30 min. Wash for 3 × 5 min, and finally seal the slides with anti-fluorescence quencher, and observe the slides with a fluorescence microscope. After viewing, the slides can be stored in a -20°C refrigerator.
如图2所示，通过对比，对比例1中传统液氮法制作切片会引起睾丸组织细胞变形，各类细胞无法辨认，无法观察睾丸形态和细胞核，本发明实施例1通过固定法制备的马睾丸冰冻切片是更加适合的方案，其可以有效地呈现马睾丸组织细胞的形态与结构。As shown in Figure 2, by contrast, in Comparative Example 1, the traditional liquid nitrogen method for making slices will cause deformation of testicular tissue cells, various types of cells cannot be identified, and testicular morphology and cell nuclei cannot be observed. Testicular frozen section is a more suitable solution, which can effectively represent the morphology and structure of equine testicular tissue cells.
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.