CN107860632B - Automatic immunohistochemical device of fruit bat wing bud - Google Patents

Abstract

Translated fromChinese

一种果蝇翅芽自动免疫组化装置，涉及生物组织抗体或荧光染料染色设备。本发明解决现有果蝇翅芽免疫组化实验人工操作，耗时费力的技术问题。本发明包括载玻片、盖玻片、一抗储液罐、二抗储液罐、PBT缓冲溶液储液罐、甲醛固定液储液罐、进液管、出液管、若干连接管和废液罐；所述载玻片设有矩形培养腔，所述盖玻片设在矩形培养腔上方，所述矩形培养腔一侧侧壁设有若干培养腔进液口，所述矩形培养腔另一侧侧壁设有若干培养腔出液口。本发明采用特制的载玻片，适用于较薄生物组织的免疫组化染色实验，可以自动完成免疫组化流程，精准地控制每一步染色和洗涤的时间，节省时间和精力，保证了实验的质量。

An automatic immunohistochemical device for Drosophila wing buds relates to biological tissue antibody or fluorescent dye staining equipment. The invention solves the technical problems of manual operation and time-consuming and laborious operation of the existing fruit fly wing bud immunohistochemical experiment. The invention includes a glass slide, a cover glass, a primary antibody storage tank, a secondary antibody storage tank, a PBT buffer solution storage tank, a formaldehyde fixative liquid storage tank, a liquid inlet pipe, a liquid outlet pipe, several connecting pipes and waste liquid tank; the glass slide is provided with a rectangular culture cavity, the cover glass is provided above the rectangular culture cavity, and one side wall of the rectangular culture cavity is provided with a plurality of culture cavity liquid inlets, and the rectangular culture cavity is provided on the other side. One side wall is provided with several culture chamber liquid outlets. The invention adopts a special glass slide, which is suitable for immunohistochemical staining experiments of thin biological tissues, can automatically complete the immunohistochemical process, precisely controls the time of each step of staining and washing, saves time and energy, and ensures the accuracy of the experiment. quality.

Description

Translated fromChinese

一种果蝇翅芽自动免疫组化装置An automated immunohistochemical device for Drosophila wing buds

技术领域technical field

本发明属于生物组织培养抗体或荧光染料染色设备技术领域，特别涉及一种果蝇翅芽自动免疫组化装置。The invention belongs to the technical field of biological tissue culture antibody or fluorescent dye staining equipment, and particularly relates to an automatic immunohistochemical device for fruit fly wing buds.

背景技术Background technique

免疫组化，是应用免疫学基本原理——抗原抗体反应，即抗原与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂荧光素、酶、金属离子、同位素显色来确定组织细胞内抗原多肽和蛋白质，对其进行定位、定性及定量的研究。Immunohistochemistry is the application of the basic principle of immunology-antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody, through chemical reaction, the colorant fluorescein, enzyme, metal ion, and isotope that label the antibody are colored to determine tissue. Intracellular antigenic polypeptides and proteins, and their localization, qualitative and quantitative research.

果蝇翅芽的免疫组化实验包括很多步骤，主要步骤有：将果蝇幼虫解剖，翅芽组织在甲醛固定液中固定40～60分钟，然后洗固定液1个小时；再将组织置于一定浓度的一抗中，在低温(4～6℃)下孵育8～12小时，然后洗涤一抗；加入二抗染色1小时，洗涤二抗。整个流程不仅步骤繁琐，而且每隔一定的时间，需要人工加样和换洗液体，耗费时间和精力，如果某些步骤错过加样时间或是染色时间过长，都会影响最终的免疫组化效果。The immunohistochemical experiment of Drosophila wing buds includes many steps. The main steps are: dissect the Drosophila larvae, fix the wing bud tissue in formaldehyde fixative for 40 to 60 minutes, and then wash the fixative for 1 hour; In a certain concentration of primary antibody, incubate at low temperature (4-6°C) for 8-12 hours, and then wash the primary antibody; add secondary antibody for staining for 1 hour, and wash the secondary antibody. The whole process is not only cumbersome, but also needs to manually add samples and change the washing liquid at regular intervals, which consumes time and energy. If some steps are missed in the sample addition time or the staining time is too long, the final immunohistochemical effect will be affected.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于克服现有技术的缺点，提供一种果蝇翅芽自动免疫组化装置，解决现有果蝇翅芽免疫组化实验人工操作，耗时费力的技术问题。The purpose of the present invention is to overcome the shortcomings of the prior art, provide an automatic immunohistochemical device for Drosophila wing buds, and solve the technical problems of manual operation, time-consuming and laborious in the existing Drosophila wing bud immunohistochemical experiments.

本发明是通过以下技术方案实现的：The present invention is achieved through the following technical solutions:

一种果蝇翅芽自动免疫组化装置，其中：包括载玻片、盖玻片、一抗储液罐、二抗储液罐、PBT缓冲溶液储液罐、甲醛固定液储液罐、进液管、出液管、若干连接管和废液罐；A Drosophila wing bud automatic immunohistochemical device, comprising: a glass slide, a cover glass, a primary antibody storage tank, a secondary antibody storage tank, a PBT buffer solution storage tank, a formaldehyde fixative liquid storage tank, an inlet Liquid pipe, liquid outlet pipe, several connecting pipes and waste liquid tank;

所述载玻片设有矩形孵育腔，所述盖玻片设在矩形孵育腔上方，所述矩形孵育腔一侧侧壁上设有若干孵育腔进液口，所述矩形孵育腔另一侧侧壁上设有若干孵育腔出液口；The glass slide is provided with a rectangular incubation cavity, the cover glass is provided above the rectangular incubation cavity, a number of incubation cavity liquid inlets are arranged on one side wall of the rectangular incubation cavity, and the other side of the rectangular incubation cavity is provided A number of incubation chamber liquid outlets are arranged on the side wall;

所述一抗储液罐、二抗储液罐、PBT缓冲溶液储液罐和甲醛固定液储液罐设在载玻片一侧上方，所述废液罐设在载玻片另一侧下方，所述一抗储液罐、二抗储液罐、PBT缓冲溶液储液罐、甲醛固定液储液罐的出口分别通过连接管与进液管连接，所述进液管通过若干孵育腔进液口与矩形孵育腔连接；所述若干孵育腔出液口通过出液管与废液罐连接；The primary antibody storage tank, the secondary antibody storage tank, the PBT buffer solution storage tank and the formaldehyde fixative liquid storage tank are arranged above one side of the glass slide, and the waste liquid tank is arranged under the other side of the glass slide , the outlets of the primary antibody storage tank, the secondary antibody storage tank, the PBT buffer solution storage tank, and the formaldehyde fixative liquid storage tank are respectively connected with the liquid inlet pipe through a connecting pipe, and the liquid inlet pipe enters the liquid inlet pipe through several incubation chambers. The liquid port is connected with the rectangular incubation chamber; the liquid outlet ports of the several incubation chambers are connected with the waste liquid tank through the liquid outlet pipe;

所述一抗储液罐、二抗储液罐、PBT缓冲溶液储液罐、甲醛固定液储液罐与进液管之间的连接管上均设有由单片机控制模块控制的微型液体控制阀，所述出液管上设有由单片机控制模块控制的抽液泵；The connecting pipes between the primary antibody liquid storage tank, the secondary antibody liquid storage tank, the PBT buffer solution liquid storage tank, the formaldehyde fixed solution liquid storage tank and the liquid inlet pipe are all provided with micro-liquid control valves controlled by the single-chip control module. , the liquid outlet pipe is provided with a liquid pump controlled by a single chip control module;

所述一抗储液罐、二抗储液罐以及孵育腔上设有由单片机控制模块控制的温度控制装置，所述载玻片上设有检测矩形孵育腔温度的红外测温探头。The primary antibody liquid storage tank, the secondary antibody liquid storage tank and the incubation chamber are provided with a temperature control device controlled by a single-chip control module, and the glass slide is provided with an infrared temperature probe for detecting the temperature of the rectangular incubation chamber.

进一步，所述孵育腔进液口有2～5个；所述孵育腔出液口与孵育腔进液口的数量一致。Further, there are 2 to 5 liquid inlets in the incubation chamber; the number of liquid outlets in the incubation chamber is consistent with the number of liquid inlets in the incubation chamber.

本发明中的盖玻片可实现矩形孵育腔的密封，避免外界微生物侵染孵育液；所述一抗储液罐、二抗储液罐、PBT缓冲溶液储液罐、甲醛固定液储液罐、连接管、微型液体控制阀、进液管、出液管和废液罐的设置，可根据需要对矩形孵育腔内的孵育液进行更换，特别是在甲醛固定液、一抗、二抗、PBT缓冲溶液、储液罐与进液管之间设置了微型液体控制阀，并在出液管上置了抽液泵，可实现自动操作；一抗、二抗储液罐上低温控制装置的设置，载玻片上设有红外测温探头，确保翅芽组织一抗染色时在低温下孵育。The cover glass in the present invention can realize the sealing of the rectangular incubation cavity to prevent the incubation liquid from being infected by external microorganisms; the primary antibody storage tank, the secondary antibody storage tank, the PBT buffer solution storage tank, and the formaldehyde fixative liquid storage tank , connecting pipe, micro liquid control valve, liquid inlet pipe, liquid outlet pipe and waste liquid tank settings, the incubation solution in the rectangular incubation chamber can be replaced as needed, especially in the formaldehyde fixative, primary antibody, secondary antibody, A micro-liquid control valve is set between the PBT buffer solution, the liquid storage tank and the liquid inlet pipe, and a liquid pump is set on the liquid outlet pipe, which can realize automatic operation; the low temperature control device on the primary antibody and secondary antibody liquid storage tanks Setting, there is an infrared thermometer probe on the slide to ensure that the wing bud tissue is incubated at a low temperature when the primary antibody is stained.

本发明采用特制的载玻片，适用于较薄生物组织的免疫组化染色实验，可以自动完成免疫组化流程，精准地控制每一步染色和洗涤的时间，节省时间和精力，保证了实验的质量。The invention adopts a special glass slide, which is suitable for immunohistochemical staining experiments of thin biological tissues, can automatically complete the immunohistochemical process, precisely controls the time of each step of staining and washing, saves time and energy, and ensures the accuracy of the experiment. quality.

附图说明Description of drawings

图1是一种果蝇翅芽自动免疫组化装置的结构示意图；Fig. 1 is the structural representation of a Drosophila wing bud automatic immunohistochemical device;

图2是载玻片、盖玻片的结构示意图；Fig. 2 is the structural representation of slide glass and cover glass;

图3是图2的俯视图；Fig. 3 is the top view of Fig. 2;

图4是单片机控制电路原理框图。Figure 4 is a schematic block diagram of the microcontroller control circuit.

具体实施方式Detailed ways

下面结合附图和实施例对本发明作进一步的详细描述。The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

如图1至图3所示，本实施例中的一种果蝇翅芽自动免疫组化装置，包括载玻片1、盖玻片2、一抗储液罐10、二抗储液罐11、PBT缓冲溶液储液罐12、甲醛固定液储液罐13、进液管4、出液管5、若干连接管3和废液罐6；As shown in FIGS. 1 to 3 , a Drosophila wing bud automatic immunohistochemical device in this embodiment includes aglass slide 1 , acover glass 2 , a primaryantibody storage tank 10 , and a secondaryantibody storage tank 11 , PBT buffersolution storage tank 12, formaldehyde fixedliquid storage tank 13, liquid inlet pipe 4,liquid outlet pipe 5, several connectingpipes 3 and waste liquid tank 6;

所述载玻片1设有矩形孵育腔7，所述盖玻片2设在矩形孵育腔7上方，所述矩形孵育腔7一侧侧壁上设有若干孵育腔进液口8，所述矩形孵育腔7另一侧侧壁上设有若干孵育腔出液口9；Theslide 1 is provided with arectangular incubation cavity 7 , thecover glass 2 is provided above therectangular incubation cavity 7 , and a number of incubation cavity liquid inlets 8 are provided on one side wall of therectangular incubation cavity 7 . The other side wall of therectangular incubation chamber 7 is provided with several incubation chamber liquid outlets 9;

所述一抗储液罐10、二抗储液罐11、PBT缓冲溶液储液罐12和甲醛固定液储液罐13设在载玻片1一侧上方，所述废液罐6设在载玻片1另一侧下方，所述一抗储液罐10、二抗储液罐11、PBT缓冲溶液储液罐12、甲醛固定液储液罐13的出口分别通过连接管3与进液管4连接，所述进液管4通过若干孵育腔进液口8与矩形孵育腔7连接；所述若干孵育腔出液口9通过出液管5与废液罐6连接；The primaryantibody storage tank 10, the secondaryantibody storage tank 11, the PBT buffersolution storage tank 12 and the formaldehyde fixativeliquid storage tank 13 are arranged above one side of theslide glass 1, and the waste liquid tank 6 is arranged on the carrier. Below the other side of theslide 1, the outlets of the primaryantibody storage tank 10, the secondaryantibody storage tank 11, the PBT buffersolution storage tank 12, and the formaldehyde fixativeliquid storage tank 13 pass through the connectingpipe 3 and the liquid inlet pipe respectively. 4 is connected, the liquid inlet pipe 4 is connected to therectangular incubation chamber 7 through several incubation chamber liquid inlets 8; the several incubation chamber liquid outlets 9 are connected to the waste liquid tank 6 through theliquid outlet pipe 5;

所述一抗储液罐10、二抗储液罐11、PBT缓冲溶液储液罐12、甲醛固定液储液罐13与进液管4之间的连接管3上均设有由单片机控制模块控制的微型液体控制阀14，所述出液管5上设有由单片机控制模块控制的抽液泵15。The primary antibodyliquid storage tank 10, the secondary antibodyliquid storage tank 11, the PBT buffer solutionliquid storage tank 12, the formaldehyde fixedliquid storage tank 13 and the connectingpipe 3 between the liquid inlet pipe 4 are all provided with a single-chip control module. Themicro-liquid control valve 14 is controlled, and theliquid outlet pipe 5 is provided with aliquid pump 15 controlled by the single-chip control module.

所述一抗储液罐、二抗储液罐上设有由单片机控制模块控制的低温控制装置，所述载玻片1上设有红外测温探头16，检测矩形孵育腔7的温度。The primary antibody liquid storage tank and the secondary antibody liquid storage tank are provided with a low temperature control device controlled by a single-chip control module, and an infraredtemperature measuring probe 16 is provided on theglass slide 1 to detect the temperature of therectangular incubation chamber 7 .

所述孵育腔进液口有2～5个(本实施例为2个)；所述孵育腔出液口与孵育腔进液口的数量一致。The incubation cavity has 2 to 5 liquid inlets (two in this embodiment); the incubation cavity liquid outlet and the incubation cavity have the same number of liquid inlets.

如图4所示，所述一抗储液罐10、二抗储液罐11、PBT缓冲溶液储液罐12、甲醛固定液储液罐13与进液管4之间的连接管3上设置的微型液体控制阀14和抽液泵15，均与单片机控制模块连接，从而实现操作自动化。As shown in FIG. 4 , theconnection pipe 3 between the primaryantibody storage tank 10 , the secondaryantibody storage tank 11 , the PBT buffersolution storage tank 12 , the formaldehyde fixativeliquid storage tank 13 and the liquid inlet pipe 4 is provided with Themicro-liquid control valve 14 and theliquid pump 15 are all connected with the single-chip control module, so as to realize the operation automation.

如图4所示，所述红外测温探头16与单片机控制模块连接，实时测量载玻片孵育腔内的温度，所述一抗储液罐10、二抗储液罐11的低温控制装置与单片机控制模块连接，红外测温探头所测数据反馈给单片机，单片机再控制一抗储液罐10、二抗储液罐11的低温控制装置，从而确保在一抗溶液孵育时，孵育腔的温度为4～6℃，其他染色步骤，孵育腔的温度均控制在室温。As shown in FIG. 4 , the infraredtemperature measuring probe 16 is connected to the single-chip control module to measure the temperature in the incubation chamber of the slide in real time. The low temperature control devices of the primaryantibody storage tank 10 and the secondaryantibody storage tank 11 are connected to The single-chip microcomputer control module is connected, the data measured by the infrared temperature measuring probe is fed back to the single-chip computer, and the single-chip microcomputer controls the low-temperature control devices of the primary antibodyliquid storage tank 10 and the secondary antibodyliquid storage tank 11, so as to ensure the temperature of the incubation chamber when the primary antibody solution is incubated. For other staining steps, the temperature of the incubation chamber was controlled at room temperature.

本发明的使用过程：The use process of the present invention:

使用时，将在磷酸缓冲液中解剖下来的翅芽转移到矩形孵育腔7内，盖上盖玻片2，并用中性树脂或凡士林将盖玻片2四周密封；When in use, transfer the dissected wing buds in the phosphate buffer into therectangular incubation chamber 7, cover with acover glass 2, and seal thecover glass 2 around with neutral resin or Vaseline;

通过单片机控制电路：Control circuit through microcontroller:

首先打开甲醛固定液储液罐13下方连接管上的微型液体控制阀14和抽液泵15，确保载玻片储液槽充满固定液后，关闭甲醛固定液储液罐13下方连接管上的微型液体控制阀14和抽液泵15，固定40～60min；First, open themicro-liquid control valve 14 and thepump 15 on the connecting pipe below the formaldehyde fixativeliquid storage tank 13 to ensure that the slide liquid storage tank is full of fixative, and then close the connecting pipe under the formaldehyde fixativeliquid storage tank 13. Themicro-liquid control valve 14 and thepump 15 are fixed for 40-60 minutes;

然后，开启PBT缓冲溶液储液罐12下方连接管上的微型液体控制阀14和抽液泵15，缓冲液持续清洗甲醛固定液1～1.5h后，关闭PBT缓冲溶液储液罐12下方连接管上的微型液体控制阀14和抽液泵15；Then, open themicro-liquid control valve 14 and thepump 15 on the connecting pipe below the PBT buffersolution storage tank 12 , and after the buffer continues to wash the formaldehyde fixative solution for 1 to 1.5 hours, close the connecting pipe under the PBT buffersolution storage tank 12 Microliquid control valve 14 andpump 15 on the

同时开启一抗储液罐10下方连接管上的微型液体控制阀14和抽液泵15，确保一抗低温溶液充满载玻片储液槽并充分浸泡翅芽组织后，关闭一抗储液罐10下方连接管上的微型液体控制阀14和抽液泵15，低温孵育8～12h。At the same time, open themicro-liquid control valve 14 and thepump 15 on the connecting pipe below the primaryantibody storage tank 10 to ensure that the primary antibody low-temperature solution fills the slide storage tank and fully soaks the wing bud tissue, and then closes the primary antibody storage tank. 10 Connect themicro-liquid control valve 14 and thepump 15 on the lower part of the tube, and incubate at low temperature for 8-12 hours.

再次开启PBT缓冲溶液储液罐12下方连接管上的微型液体控制阀14和抽液泵15，缓冲液持续清洗一抗溶液1～1.5h后，关闭PBT缓冲溶液储液罐12下方连接管上的微型液体控制阀14和抽液泵15；Open themicro-liquid control valve 14 and thepump 15 on the connecting pipe below the PBT buffersolution storage tank 12 again. After the buffer continues to wash the primary antibody solution for 1 to 1.5 hours, close the connecting pipe under the PBT buffersolution storage tank 12. The microliquid control valve 14 and theliquid pump 15;

同时开启二抗储液罐11下方连接管上的微型液体控制阀14和出液管5上的抽液泵15，确保二抗低温溶液充满载玻片储液槽并充分浸泡翅芽组织后，关闭二抗储液罐10下方连接管上的微型液体控制阀14和抽液泵15，低温孵育1～1.2h。At the same time, open themicro-liquid control valve 14 on the connecting pipe below the secondaryantibody storage tank 11 and theliquid pump 15 on theliquid outlet pipe 5 to ensure that the secondary antibody low-temperature solution fills the slide storage tank and fully soaks the wing bud tissue. Close themicro-liquid control valve 14 and thepump 15 on the connecting pipe below the secondaryantibody storage tank 10, and incubate at low temperature for 1-1.2 hours.

最后开启PBT缓冲溶液储液罐12下方连接管上的微型液体控制阀14和出液管5上的抽液泵15，缓冲液持续清洗二抗溶液1～1.5h后，关闭PBT缓冲溶液储液罐12下方连接管上的微型液体控制阀14和抽液泵15，完成实验。Finally, turn on themicro-liquid control valve 14 on the connecting pipe below the PBT buffersolution storage tank 12 and theliquid pump 15 on theliquid outlet pipe 5. After the buffer solution continues to clean the secondary antibody solution for 1 to 1.5 hours, the PBT buffer solution storage solution is closed. Connect themicro-liquid control valve 14 on the pipe below thetank 12 and thepump 15 to complete the experiment.

实验完成后，将载玻片直接放置荧光显微镜或激光共聚焦显微镜下拍照。After the experiment was completed, the slides were directly placed under a fluorescence microscope or laser confocal microscope to take pictures.

所述盖玻片在盖住孵育腔，并用中性树脂等粘合剂密封四周之后，孵育腔，孵育腔进液口，孵育腔出液口以及相连的进液管，出液管形成密闭的空间，以便抽液泵可以将孵育腔内的液体抽走。After the cover glass covers the incubation chamber and seals it with an adhesive such as neutral resin for four weeks, the incubation chamber, the liquid inlet of the incubation chamber, the liquid outlet of the incubation chamber and the connected liquid inlet pipe form a closed liquid outlet. space so that the liquid in the incubation chamber can be removed by the aspiration pump.

本发明能够以多种形式具体实施而不脱离发明的精神或实质，所以应当理解，上述实施例不限于前述的细节，而应在权利要求所限定的范围内广泛地解释，因此落入权利要求或其等效范围内的变化和改型都应为权利要求所涵盖。The present invention can be embodied in various forms without departing from the spirit or essence of the invention, so it should be understood that the above-described embodiments are not limited to the foregoing details, but should be construed broadly within the scope defined by the claims, and thus fall within the claims Changes and modifications within the scope of their equivalents should be covered by the claims.

Claims (2)

1. The utility model provides an automatic immunohistochemical device of fruit bat wing bud which characterized in that: comprises a glass slide (1), a cover glass (2), a primary antibody liquid storage tank (10), a secondary antibody liquid storage tank (11), a PBT buffer solution liquid storage tank (12), a formaldehyde stationary liquid storage tank (13), a liquid inlet pipe (4), a liquid outlet pipe (5), a plurality of connecting pipes (3) and a waste liquid tank (6);

the glass slide (1) is provided with a rectangular incubation cavity (7), the cover glass (2) is arranged above the rectangular incubation cavity (7), a plurality of incubation cavity liquid inlets (8) are formed in the side wall of one side of the rectangular incubation cavity (7), and a plurality of incubation cavity liquid outlets (9) are formed in the side wall of the other side of the rectangular incubation cavity (7);

the first anti-liquid storage tank (10), the second antibody liquid storage tank (11), the PBT buffer solution liquid storage tank (12) and the formaldehyde fixing solution liquid storage tank (13) are arranged above one side of the glass slide (1), the waste liquid tank (6) is arranged below the other side of the glass slide (1), outlets of the first anti-liquid storage tank (10), the second antibody liquid storage tank (11), the PBT buffer solution liquid storage tank (12) and the formaldehyde fixing solution liquid storage tank (13) are respectively connected with the liquid inlet pipe (4) through connecting pipes (3), and the liquid inlet pipe (4) is connected with the rectangular incubation cavity (7) through a plurality of incubation cavity liquid inlets (8); the liquid outlets (9) of the incubation cavities are connected with a waste liquid tank (6) through a liquid outlet pipe (5);

a micro liquid control valve (14) controlled by a singlechip control module is arranged on each connecting pipe (3) between the first anti-liquid storage tank (10), the second anti-liquid storage tank (11), the PBT buffer solution storage tank (12), the formaldehyde fixing liquid storage tank (13) and the liquid inlet pipe (4), and a liquid pump (15) controlled by the singlechip control module is arranged on the liquid outlet pipe (5);

the first anti-liquid storage tank (10) and the second anti-liquid storage tank (12) are provided with low-temperature control devices controlled by the singlechip control module，An infrared temperature measuring probe (16) for detecting the temperature of the rectangular incubation cavity (7) is arranged on the glass slide (1).

2. The automatic immunohistochemical device of fruit fly wing bud according to claim 1, characterized in that: 2-5 liquid inlets (8) of the incubation cavity are provided; the number of the incubation cavity liquid outlets (9) is consistent with that of the incubation cavity liquid inlets (8).

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