做dry lab的读文章时看到各种实验技术老是一头雾水，非常有必要科普下那些经常出现在paper里的实验技术。
dry lab要想做得好，必须要精通实验技术，了解他们的核心需求！！！
Wnt1-Cre transgenic mouse model
lineage tracing by Cre-LoxP system - A step by step discussion
什么是Cre？
recombinase enzyme isolated from bacteriophage P1 which targets LoxP DNA sequences for the process of recombination
In the Cre mouse every cell contains the coding sequence of Cre, but only cells expression protein X will express the Cre
In the reporter mouse every cell contains the coding sequence of GFP, but none of them give GFP signal because of presence of STOP sequence.
heterozygous mouse
Once the stop sequence is removed from stem cell DNA then every cell originated from stem cell will give positive GFP signal.
从而达到lineage tracing的效果。
Wnt1Cre/R26RTomato mouse embryos - 为什么用这个老鼠？
ROSA26 is a locus used for constitutive, ubiquitous gene expression in mice. It was first isolated in 1991 in a gene-trap mutagenesis screen of embryonic stem cells (ESCs). Over 130 knock-in lines have been created based on the ROSA26 locus.
Wnt1 - 为什么用这个基因？
Wnt1Cre recombines in the dorsal neural tube where the premigratory neural crest resides.
用这个技术标定了这个细胞家系（premigratory neural crest），然后用单细胞技术measured mRNAs of single TOMATO+ cells
asynchrony - 异步的：与同步相反，不在同一时间发生。
anteroposterior axis of the embryology，通俗点就是从头到尾。
dorsal to ventral，从背到腹部。
epithelial-to-mesenchymal transition (EMT) 上皮到间叶细胞的转换。
Immunohistochemistry - 发育生物学的主要利器，拍片。
Immunohistochemistry - YouTube
根据感兴趣的分子（即抗原，antigen），找对应抗体，一级抗体负责找到我们的antigen，然后加入二级抗体（包含了一个结合抗体和一个催化酶，如HRP酶），最终加入DAB，DAB被局部催化反应形成带颜色的复合物，从而被显微镜观察到。
In the schematic illustration (Figure 1) a formalin-fixed paraffin embedded tissue section is stained using a primary antibody directed towards a specific protein target. A solution containing the primary antibody is added to the tissue section and the antibodies are allowed some time to find and bind to their target. After this step, unbound and surplus antibodies are washed away and the secondary antibody is added. The secondary antibody, which carries a linker molecule with horseradish peroxidase (HRP) enzymes, is also allowed some time to bind to the primary antibody, followed by another washing step. After this, 3,3' Diaminobenzidine (DAB) is added. The HRP enzyme transforms the DAB substrate into a brownish precipitate that is deposited in the tissue at the site of the reaction, thus producing a visual representation of where the primary antibody first bound its target.
注：epitopes即是蛋白上的某个部分，相对于抗原。
看一个案例：
A是一个几毫米的图，里面只有两种颜色，红色就是转基因小鼠的颜色，绿色就是SOX10蛋白的颜色；这张图看着立体，是因为扫描了很多层然后通过计算机合成的。SOX10+/Wnt1TOM+标记的就是neural crest cells (NCCs)
B是A里面的切片部分的截面图，大概有三种颜色，红色就是转基因小鼠的颜色，绿色就是SOX10蛋白的颜色，黄色就是都表达的部分。展现了cranial and trunk portions的分离。
目的：表面一个核心的生物学过程，NCCs在头部和躯干里迁移，
荧光蛋白的选择：
GFP：绿色的优先级最高，灵敏、剂量需求小
Tomato：红色
YFP：黄色
具体操作时，每一个蛋白都有自己独特的光谱，可以自由的设定颜色，但是通常都会选择红、绿、蓝。
7 fluorescent labels：
DAPI：标记细胞核
In situ sequencing - 原位测序
spatial single cell sequencing，单细胞级别的测序，通过barcode杂交，得到扩增的指定基因的序列，然后再用荧光去杂交，最终可以在显微镜下看到每一个细胞的基因表达。（指定RNA扩增 和 荧光显微 技术的完美结合）
mouse lineage
Immunohistochemistry of control Neurog2CreERT2/+;R26RYFP/+ (carrying one copy of CreERT2) and mutant Neurog2CreERT2/CreERT2;R26RYFP/+ (carrying two copies of CreERT2) tamoxifen-injected at E9.5 and analyzed at E15.5
上标表示Neurog2基因被CreERT2给替换了，两个上标都是CreERT2则表示Neurog2的两个allele都被替换了，也就是给knock out了。
+：wild type
-：knock out
YFP：插入或替换了YFP
一个案例：
参考：