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.
Fixation
Immunofluorescence
75% MeOH/H2O | 10min |
50% MeOH/PBS | 10min |
25% MeOH/PBS | 10min |
PBS | 10min |
PBS | 10min |
30% EtOH/H2O | 10 min |
50% EtOH/H2O | 10 min |
75% EtOH/H2O | 10 min |
100% EtOH | 10 min |
Infiltration
Embedding
Sectioning
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 |