搜索

Barrier Function Assay
屏障功能试验   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

This protocol serves to determine the integrity of the barrier function of (murine) epidermis. Defects in the barrier function lead to dehydration and infection in neonatal mice/humans. One possible way to assess epidermal barrier integrity is by a dye penetration assay as described hereunder. This assay should be done on unfixed, untreated tissues (e.g. formaldehyde- or glutaraldehyde-fixed) or on whole mouse embryos (E18.5). This protocol was adapted from Hardman (1998).

Keywords: Skin integrity(皮肤的完整性), Permiability(渗), Dye penetration assay(染料渗透法)

Materials and Reagents

  1. E18.5 embryos from mice
  2. Chilled methanol in water (25%, 50%, 75% and 100% MetOH)
  3. 0.1% toluidine blue (in water) (Sigma-Aldrich)
  4. Chilled 1x PBS (pH 7.4) (Sigma-Aldrich, catalog number: P4417 ) (see Recipes)

Equipment

  1. Forceps
  2. Camera

Procedure

  1. Isolate uterus from pregnant females to obtain E18.5 embryos.
  2. Euthanize embryos by immersion in ice cold PBS for 30 min.
  3. At 18.5 days gestation, cull mother by cervical dislocation. Make a ventral incision and remove the uterine horn containing the embryos. To euthanize embryos, immerse entire uterus containing embryos in ice cold PBS for 30 min. Separate embryos and remove from uterus. Check that embryos have been successfully euthanized by pinching toe or tail with forceps. If embryo responds, place back into ice cold PBS and check every 5 min. Once embryos are unresponsive/culled, proceed to the next step.
  4. Finger/toe clip individual embryos for identification. This is especially important when crossing heterozygous mice. The digits can be used for genotyping the embryos.
  5. Passage embryos through chilled methanol gradient, taking 2 min per step. It is crucial to have all solutions at 4 degrees centigrade. Keep solutions on ice throughout protocol.
    1. 25% methanol in water
    2. 50% methanol in water
    3. 75% methanol in water
    4. 100% methanol
    5. 75% methanol in water
    6. 50% methanol in water
    7. 25% methanol in water
    8. 100% water or PBS
  6. Immerse embryos in 0.1% toluidine blue solution in water for 1-2 min on ice.
  7. Destain embryos in PBS (pH 7.4) until a dye pattern emerges (in case of barrier defect) or until dye is washed away (in case of intact barrier) on ice.
  8. Take pictures of individual embryos and if relevant genotype embryos. See also Figure 1 for some examples of intact barriers and barrier acquisition defects.


    Figure 1. Examples of intact barriers and barrier acquisition defects

Recipes

  1. 1x PBS (pH 7.4)
    1 tablet per 200 ml dH2O

Acknowledgments

This protocol was adapted from Hardman et al. (1998). Our work is supported by the Dutch Cancer Society (RUG 2012-5549), Stichting Kinder Oncologie Groningen (SKOG) and EMBO grants to FF.

References

  1. Hardman, M. J., Sisi, P., Banbury, D. N. and Byrne, C. (1998). Patterned acquisition of skin barrier function during development. Development 125(8): 1541-1552.

简介

该方案用于确定(鼠)表皮的屏障功能的完整性。 屏障功能中的缺陷导致新生小鼠/人中的脱水和感染。 评估表皮屏障完整性的一种可能方式是通过如下所述的染料渗透测定。 该测定应该在未固定的,未处理的组织(例如固定的甲醛或戊二醛)或整个小鼠胚胎(E18.5)上进行。 该协议改编自Hardman(1998)。

关键字:皮肤的完整性, 渗, 染料渗透法

材料和试剂

  1. E18.5小鼠胚胎
  2. 冷冻甲醇的水(25%,50%,75%和100%甲醇)
  3. 0.1%甲苯胺蓝(在水中)(Sigma-Aldrich)
  4. 冷冻1×PBS(pH7.4)(Sigma-Aldrich,目录号:P4417)(参见Recipes)

设备

  1. 镊子
  2. 相机

程序

  1. 隔离怀孕女性的子宫,以获得E18.5胚胎
  2. 通过浸泡在冰冷的PBS中30分钟安乐死胚胎。
  3. 在妊娠18.5天,通过颈椎脱位将母亲剔除。做腹部切口,并取出含有胚胎的子宫角。为了安乐死胚胎,将整个含有胚胎的子宫浸入冰冷的PBS中30分钟。分离胚胎和从子宫中删除。检查胚胎已经成功安乐死用镊子夹住脚趾或尾巴。如果胚胎反应,放回冰冷的PBS中,每5分钟检查一次。一旦胚胎无反应/剔除,进行下一步。
  4. 手指/脚趾剪切单个胚胎进行识别。这在杂交小鼠杂交时特别重要。数字可用于对胚胎进行基因分型。
  5. 通过冷冻甲醇梯度通道胚胎,每步2分钟。所有的解决方案在4摄氏度是至关重要的。保持解决方案在冰整个协议。
    1. 25%甲醇/水
    2. 50%甲醇的水溶液
    3. 75%甲醇的水溶液
    4. 100%甲醇
    5. 75%甲醇的水溶液
    6. 50%甲醇的水溶液
    7. 25%甲醇/水
    8. 100%水或PBS
  6. 浸泡在0.1%甲苯胺蓝色溶液中的胚胎在水中,在冰上1-2分钟
  7. 在PBS(pH 7.4)中脱胚直至出现染料图案(在屏障缺陷的情况下)或直到在冰上洗掉染料(在完整屏障的情况下)。
  8. 拍摄个体胚胎和相关基因型胚胎的照片。 参见图1,了解完整障碍和障碍获取缺陷的一些例子

    图1。 完整障碍和障碍获取缺陷的示例

食谱

  1. 1x PBS(pH 7.4)
    每200ml dH 2 O 1片剂1片

致谢

该方案改编自Hardman等人(1998)。 我们的工作由荷兰癌症协会(RUG 2012-5549),Stichting Kinder Oncologie Groningen(SKOG)和EMBO授予FF支持。

参考文献

  1. Hardman,M.J.,Sisi,P.,Banbury,D.N.and Byrne,C。(1998)。 在开发过程中对皮肤屏障功能进行图案化采集。 开发 125(8):1541-1552
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:DiTommaso, T. and Foijer, F. (2014). Barrier Function Assay. Bio-protocol 4(10): e1133. DOI: 10.21769/BioProtoc.1133.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。