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小鼠脑组织振动切片封固

郑茗芳 |  2022-11-23  | DOI: 10.21769/BioProtoc.v1114
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视频简介对小鼠脑切片进行免疫荧光染色和成像,是对特定基因的蛋白产物进行检测的常用方法,也常被应用于识别和标记脑组织中的不同细胞类型,是神经环路发育、连接和功能研究的必备技术之一。免疫荧光染色敏感性强、操作简单,但也存在染色后切片保存时间短、荧光强度随时间延长而逐渐变弱的问题[1]。 为延长保存时间,在免疫染色之后通常需要进行封片处理。封片是将切片封固保存于载玻片与盖玻片之间的操作,可使切片不与空气接触,防止其脱水、氧化;使用抗荧光淬灭封片剂还可减缓荧光淬灭,便于镜检观察及保存[2]。本视频详细介绍了小鼠脑组织切片的封片步骤,并针对实验中常见问题给出注意事项与解决方案。 (参考文献见附件)
  • 视频介绍

一、视频摘要

对小鼠脑切片进行免疫荧光染色和成像,是对特定基因的蛋白产物进行检测的常用方法,也常被应用于识别和标记脑组织中的不同细胞类型,是神经环路发育、连接和功能研究的必备技术之一。免疫荧光染色敏感性强、操作简单,但也存在染色后切片保存时间短、荧光强度随时间延长而逐渐变弱的问题[1]。

为延长保存时间,在免疫染色之后通常需要进行封片处理。封片是将切片封固保存于载玻片与盖玻片之间的操作,可使切片不与空气接触,防止其脱水、氧化;使用抗荧光淬灭封片剂还可减缓荧光淬灭,便于镜检观察及保存[2]。本视频详细介绍了小鼠脑组织切片的封片步骤,并针对实验中常见问题给出注意事项与解决方案。


二、关键词

Mouse brain (小鼠脑);Immunofluorescence (免疫荧光);Vibratome section (振动切片)


三、实验样品信息,试剂、耗材或仪器

1.样品信息

本视频样品通过振动切片机(Leica VT1000S)制取,将固定后小鼠脑组织切为30μm冠状切片,并进行免疫荧光染色。

2.试剂和耗材

试剂:

1) 1× PBS (配方见附录)

2) Triton X-100 (sigma,X100) (25%Triton X-100配方见附录)

3) 封片剂(SouthernBiotech0100-01;LERNER LABORATORIES,13800)

4) 无色透明指甲油(国产)

耗材:

1) 培养皿

2) 48孔板(路斯特,23048) (孔径16mm,孔深17mm)

3)细毛笔(HWAHONG,345#1) (笔毛长6mm,直径1.2mm)

4) 载玻片(世态,80312-3161)

5) 盖玻片(世态,80340-3610;Fisher,12-548-B)

6) 20 μl移液枪(Eppendorf,3120000232)

7) 200 μl移液枪(Eppendorf,3120000259)

8) 无尘擦拭纸(Kimtech)

附录:

1) PBS配制

1×PBS由20×PBS稀释得到,20×PBS配方如下:

用分析天平称取表1中药品,添加800-900 ml去离子水,使用磁力搅拌器充分溶解(无任何固体),以去离子水定容至1 L并充分混匀、过滤。滤好的20×PBS可立即稀释使用;稀释得1×PBS pH=7.4。暂时不用的20×PBS分装后高压灭菌,储存于室温或4℃冰箱。

表1.配制1L 20× PBS的药品配比

2) 25%Triton配制

倾倒10 ml TritonX-100于50 ml离心管,加入30 ml去离子水或1×PBS,在旋转混匀仪上倒转混匀过夜。室温保存。

3.仪器和软件


四、实验操作

PART1 贴片Mounting

1. 在载玻片磨砂面(或白色涂料区域)写明切片信息,如实验日期、样品编号或信息(如基因型、年龄、给药情况等)、染色信息(如488Donkey@Mouse@PV、DAPI)、脑片厚度(如50μm)和玻片序号等。

Label the frosted (or white coated) end of microscopy slides with relevant information (e.g., Date; Mouse ID; Immunofluorescence staining information; Section thickness; Slide number).
2. 向培养皿中加入适量1×PBS(确保载玻片斜放于培养皿中时,能一半浸没于液面以下)。在PBS中加入微量(约10μl)25%Triton,以降低表面张力,辅助贴片。

Dump 1×PBS to a petri dish, ensuring approximately one-half area of the slide (applied in the next step) could be submerged in PBS. Lower the surface tension by addition of trace amount (approximately 10μl) of 25% surfactant Triton X-100.
3. 将载玻片斜放于培养皿。以PBS润湿载玻片。

Leave a microscopy slide half submerged by resting the labelled side at an angle on the edge of the petri dish. Rinse the slide with PBS.
4. 用细毛笔将脑片从48孔板转移到培养皿中,按顺序排布并使其吸附于载玻片上。

Use a fine-tip paintbrush to transfer brain sections from a 48 well plate to the petri dish and gently press them flat to the bottom. Mount serial slices onto the slide in the order they were sectioned.

Note:

(1)先将单片脑片平铺在液面下的载玻片上,再以毛笔尖抵住脑片一端将其拖离溶液。

For each section, first spread it flat on the slide under PBS. Hold the brush tip against one end of the sample and gently drag it out of solution.

(2)脑片刚刚脱离液面时易携带较多PBS,此时应适当停留沥去多余PBS,否则脑片即使到达预定位置也容易随液体移动。

Allow the brain section to settle on the slide briefly and drain, as PBS saturating the sample may carry it down the slide and positioned disorderly.

(3)Triton X-100可增强液体对载玻片的附着,使载玻片保持湿润。借助这层液体润滑轻轻拖动脑片(施力过大、拖动过快易造成脑片撕裂或折叠)。

Triton X-100 allows adhesion of solution to the slide surface, thus keeping the surface wet and reducing friction when samples are dragged. Gentle maneuvers are also necessary to prevent brain sections from folding or tearing.

(4)为避免先放置的脑片在贴后续脑片时过于干燥(过分干燥会导致成像背景升高、组织形态受损),在操作过程中应实时观察,及时从边缘用毛笔补充PBS,保持所有脑片在贴片完毕前保持湿润状态。

Avoid samples dried out during mounting. (It contributes to high background staining and tissue morphological injuries.) Monitor the drying and keep the sections moist by placing a few drops of PBS beside each of them as necessary.


(5)单张玻片放置多张脑片时,通常保持同一朝向且不宜过于贴近玻片边缘(否则封片时指甲油有可能覆盖在脑片上方,影响成像)。

We recommend serial sections on the same slide placed in the same orientation and not too close to the edge. (Otherwise, the nail polish applied in sealing might overlay the slices and affect imaging results.)


5. 用纸张小心吸干载玻片周围PBS,平放(或垂直)静置至玻片及脑片上方无明显液体。

Before drying, carefully remove residual PBS from edges of the mounted slide using dust-free wipes. Lay your slide horizontally (or hold it upright to allow PBS to run slowly down the slide and accelerate the evaporation process) until there is no liquid visible.

Note:

不可过分干燥(会导致成像背景升高、组织形态受损)。

As discussed in step 4, avoid over dried of brain sections.


PART2封片Sealing

1. 用移液枪吸取100μl封片剂沿玻片长轴中线均匀滴加在脑片上,避免气泡。

For each slide, pipette 100μl of mounting medium evenly along the midline without introducing any bubbles.

Note:

封片剂的用量可随盖玻片大小而调节,但注意:如果用量过多,封片剂会扩散到盖玻片表面及载玻片边缘,使指甲油无法完全封闭载玻片与盖玻片间缝隙。

The volume of the mounting medium depends on the diameter of the coverslip. It could be adjusted at will but note that spillage of mounting medium may create a breach in the subsequent sealing.


2. 盖盖玻片。

Mount coverslips onto slides.

Note:

(1)用拇指和食指固定盖玻片两侧长边使其悬空,先令盖玻片的一侧短边接触载玻片上的封片剂,再缓慢放平,使封片剂沿盖玻片与载玻片之间的缝隙延展开来,覆盖全部脑切片,直至盖玻片另一端也与载玻片贴合。

Hold a cleaned coverslip against lengthwise edges with thumb and forefinger. To avoid bubbles, gently tip one end onto the mounting medium and allow the liquid to spread uniformly over the samples as you slowly lower the rest the coverslip.


(2)若有气泡产生,小心地将盖玻片移去,用PBS冲洗载玻片3次,重复上述盖盖玻片步骤。

If bubbles are present, slowly and carefully pry the coverslip off the slide from one corner, remove the mounting medium by rinsing the mounted slide with PBS three times and then apply a new coverslip.


(3)若因加封片剂前干片不足导致组织漂浮位移,可将组织挑回PBS,充分洗净玻片上的封片剂(或换一干净玻片),重新放置、晾干、盖盖玻片。
Inadequate drying before mounting medium addition might result in sections floating and slipping. If this occurs, transfer sections into PBS and remove the mounting medium by rinsing the slide with PBS (or simply replace it with a new one). Repeat the previous mounting process.


3. 将整个玻片置于4 °C冰箱4~6h,待封片剂凝固。

Keep slides at 4°C for 4~6h and allow the mounting medium to solidify.

Note:

注意不可挤压或倾斜,避免组织变形或位移。

Take care not to press or tilt the coverslip to avoid moving or squashing of the sections.


4. 蘸取适量无色透明指甲油,沿盖玻片边缘刷一圈,完全封闭载玻片与盖玻片间缝隙,防止和盖玻片位移

Seal the coverslip by painting the edges with a rim of colorless transparent nail polish. This will avoid coverslip movement and bubble entry, meanwhile, preventing the samples from drying out for long-term preservation.

Note:

(1)蘸取指甲油应适量。蘸取过多,指甲油会扩散到盖玻片表面;蘸取过少,容易出现指甲油凝固拉丝且无法完全封闭缝隙。The amount of nail polish should be controlled, as excessive polish overflow onto the coverslip and too little amount might result in stringy and leaky sealing.

(2)完成后检查载玻片与盖玻片间是否仍存在缝隙,若有缺口应及时补充指甲油。

Observe if there is a breach and fill it up promptly.


5. 避光条件下凝固指甲油以防止荧光淬灭。

Allow the nail polish to solidify with protecting from the light (which results in fluorescence quenching).
6. 指甲油凝固后即可至显微镜下观察拍照。或将玻片置于玻片板,4 °C避光保存。

When the nail polish is dry, store the slide in a storage tray in the dark at 4 °C or proceed directly to microscopic analyses.


五、注意事项

本视频注意事项比较适于与实验操作配合阅读,因此把注意事项插在实验操作中了。

六、结果分析(可选)

七、参考文献(可选)

[1] Bienenstock, John & DOLEZEL, JOSEF. (1970). Preservation of immunofluorescence. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 18. 518. 10.1177/18.7.518.

[2] Grimwood, R. E., & Proffer, L. H. (2000). Long-term preservation of direct immunofluorescence staining in slides stored at room temperature. Journal of cutaneous pathology, 27(5), 224–227. https://doi.org/10.1034/j.1600-0560.2000.027005224.x

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