Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.
Protocol
Fixation Transfer whole embryos or dissected explants into MEMFA or 3.7% FA+PBS in a small glass scintillation vial (Fisher cat# 03-339-25B). Incubate vial on the nutator at RT, only for 2 hours, and not more than that. Do not leave the embryo in FA for more than 2 hours. Longer incubation in FA will make the embryonic tissue harder and result in a poor penetration of antibodies in the detection process. Aspirate most of FA and add Dent’s fixative up to the top of the …
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.
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).