The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
Authors
The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
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1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2University of California, Irvine (UCI)
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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).
फिक्सेशन
Immunofluorescence
| 75% MeOH / एच 2 हे | 10min |
| 50% / MeOH पीबीएस | 10min |
| 25% / MeOH पीबीएस | 10min |
| पीबीएस | 10min |
| पीबीएस | 10min |
| 30% EtOH / एच 2 हे | 10 मिनट |
| 50% EtOH / एच 2 हे | 10 मिनट |
| 75% EtOH / एच 2 हे | 10 मिनट |
| 100% EtOH | 10 मिनट |
घुसपैठ
एम्बेड
सेक्शनिंग
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| Name | Type | Company | Catalog Number | Comments |
| PBS | Buffer | 1L recipe8.0gNaCl0.2gKCl2.68gNa2HPO4Â7H2O0.2gKH2PO4The 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 4ÂC. | ||
| ÂDent’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 Scientific | 03-339-25B | |
| 0.5ml Thin-wall PCR tubes | Tool | Molecular BioProducts | 3430 | |
| Press-to-Seal Silicone Rubber Insulators | Tool | Grace Bio-Lab Inc. | 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. | ||||
So what are the advantages of plastic sectioning over frozen/wax embedding + sectioning?
1
ReplyPosted by: NishOctober 17, 2008, 11:13 AM