塑料嵌入和非洲爪蟾胚胎切片

Summary

塑料型材保持在薄薄的组织，可以用荧光抗体免疫染色的部分真正的组织形态，这种方法比较有用的，比许多类型的组织石蜡包埋或冰冻切片。染色，塑料包埋，切片的方法是在这个视频演示。

Protocol

固定在一个小玻璃闪烁瓶转移整个胚胎或解剖植（费舍尔猫＃03 – 339 – 25B）到MEMFA或3.7％FA + PBS。在室温孵育nutator小瓶， 只有 2个小时，而不是不止于此。不要离开2个多小时的胚胎在FA。在FA的潜伏期较长，会使胚胎组织的困难，并导致在检测过程中的抗体差渗透。 吸大部分的FA和添加登特的固定液的小瓶（〜4.5毫升）顶部。轻轻颠倒几次小瓶洗登特的胚胎，用?…

Materials

Material Name	Type	Company	Catalogue Number	Comment
PBS	Buffer			1L recipe 8.0g NaCl 0.2g KCl 2.68g Na2HPO4·7H2O 0.2g KH2PO4 The pH is supposed to be 7.4. Adjust the volume to 1L. Autoclave.
PBT	Buffer			Add 0.5ml of Triton X-100 and 1.0g of BSA (Molecular Biology grade, fraction V is good [>99%], but don’t use crude 97% Albumin) to 500ml of autoclaved PBS. Filtrate through 0.2mm filter cup. Store it in 4ºC.
· Dent’s fixative	Reagent			Mix 40ml of methanol and 10ml of DMSO. Keep it in –20°C.
3.7% FA+PBS	Reagent			Mix 37% formaldehyde and PBS at 1:9 ratios. Make it fresh at each time and discard the left over.
Biotinylated Anti-Mouse IgG (H+L), made in goat	Ab	Vector Laboratories	BA-9200
Biotinylated Anti-Rabbit IgG (H+L), made in goat	Ab	Vector Laboratories	BA-1000
IG’S HUADS BIOTIN GTXRBT	Ab	Invitrogen	ALI3409	BioSource™
IgG (g), Goat Anti-Mouse	Ab	Invitrogen	M30115	CALTAG™
Fluorescent streptavidin kit	Reagent	Vector Laboratories	SA-1200	Contains streptavidin labeled with three different fluorophores (FITC, Texas Red and AMCA) suitable for this procedure.
20% goat serum/PBT	Buffer			This will be used as a blocking solution.
Small paintbrush	Tool
Dull-tip dissection needle	Tool
Kulzer Technovit 7100 GMA , 3040	Reagent		7100 GMA , 3040	glycol methacrylate
Kulzer Tungsten Histoknife H	Tool	Energy Beam Sciences	H1310
75% MeOH/H2O	Buffer
50% MeOH/PBS	Buffer
25% MeOH/PBS	Buffer
30% EtOH/H2O	Buffer
50% EtOH/H2O	Buffer
75% EtOH/H2O	Buffer
100% EtOH	Buffer
Small glass scintillation vials	Tool	Fisher	03-339-25B
0.5ml Thin-wall PCR tubes	Tool	Molecular BioProducts	3430
Press-to-Seal Silicone Rubber Insulators	Tool	Grace Bio-Labs	JTR1L-2.5	32mm L x 19mm W, 2.5mm D
Thin razor blade	Tool
1.5% Agarose in PBS

Primary antibody against your antigen or epitope tags: Make an appropriate dilution in PBT. Biotin-conjugated secondary antibody: various vendors sell biotin-conjugated antibodies.