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.
Ogata, S., Kawauchi, S., Calof, A., Cho, K. W. Plastic Embedding and Sectioning of Xenopus laevis Embryos. J. Vis. Exp. (3), e188, doi:10.3791/188 (2007).