Plastic Embedding and Sectioning of Xenopus laevis Embryos

Summary

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

1. 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.
   
   
2. Aspirate most of FA and add Dent's fixative up to the top of the vial (~ 4.5ml). Gently invert the vial for a few times to wash the embryos in Dent's, exchange with another 4.5ml of fresh Dent's, and incubate it in -20ºC

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	Buffer			1 L recipe: 8.0 g NaCl 0.2 g KCl 2.68 g Na2HPO4·7H2O 0.2 g KH2PO4The pH is supposed to be 7.4. Adjust the volume to 1 L. Autoclave.
PBT	Buffer			Add 0.5 mLl of Triton X-100 and 1.0 g of BSA (Molecular Biology grade, fraction V is good [>99%], but don’t use crude 97% Albumin) to 500ml of autoclaved PBS. Filtrate through 0.2 mm filter cup. Store it in 4ºC.
·Dent’s fixative	Reagent			Mix 40 mL of methanol and 10 mL 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/H22O	Buffer
50% EtOH/H2O	Buffer
75% EtOH/H2O	Buffer
100% EtOH	Buffer
Small glass scintillation vials	Tool	Fisher Scientific	03-339-25B
0.5 mL Thin-wall PCR tubes	Tool	Molecular BioProducts	3430
Press-to-Seal Silicone Rubber Insulators	Tool	Grace Bio-Lab Inc.	JTR1L-2.5	32 mm L x 19 mm W, 2.5 mm 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.