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| Author Information | |
| Primary Author | |
| Michael Klymkowsky ( klym@spot.Colorado.EDU ) | |
| Affiliation | |
| University of Colorado, Boulder , United States | |
| Co-Author(s) | |
| Protocol Information | |
| Protocol ID / PID | |
| PUB197 | |
| Category | |
| Cell Biology | |
| Title | |
| Tissue sectioning: Cryostat sectioning method | |
| Overview | |
| (ala Fagotto & Gumbiner, 1994) | |
| Material | |
| MEMPfa: 0.1M MOPS -- pH 7.4 2mM EGTA 1mM MgSO4 (typically this is made up as 10X MSalts. 4% paraformaldehyde (diluted from a frozen stock of 20%) (store MEMPfa at 4C and use within 1 day). eDents: 80% ethanol : 20% DMSO (can be stored at r.t. forever). eDents bleach: 1 part 30% hydrogen peroxide & 2 parts eDents.MAB: 0.1 M maleic acid 0.15 M NaCl Adjust to pH 7.5 with NaOH Blocking Buffer: Dilute blocking solution into MAB | |
| Procedure | |
| fix embryos with a fixative compatible with the antibodies to be used eDent's fixative and MEMPfa are all good choices fix for 2 h at rt followed by 30 min. in eDents * Wash briefly in PBS and soak in either 15% cold water fish gelatin/15% sucrose in PBS overnight or in 7.5% porcine gelatin (300 Bloom) 15% sucrose in PBS for 6 h at 37oC. * Embed in 15% sucrose/7.5% gelatin, chill to 4oC (you can hold the samples here for a few days). * Remove sucrose/gelatin solution and add Tissue-Tek OCT (Miles Scientific, Naperville, IL 60566), compound, allow to equilibrate for at least 30 min (it can stay in there longer). * Prepare a block using Tissue-Tek. By controlling temperatures, it is then possible to position your sample on the block in the desired orientation. Typically this is done using a stereomicroscope and forceps. Once in position, re-freeze the block, either in a -80oC freezer on using a dry ice/ethanol bath. * Place the block into the cyrostat and allow it to equilibrate to the cutting temperature (i.e. -17 oC) * Prepare 12-14 mm thick sections using a cryostat at -17°C and collected onto pre-coated or frosted glass slides and store at -80oC until you are ready to stain them. (Colorfrost/Plus - Fisher Scientific Co). * Warm slides and allow them to dry at room temperature * extract for 2 min. in 100% acetone. * rehydrate in PBS * block with TNB blocking buffer (from tyramide kit) for 30 min at r.t. or just incubate in PBS + 0.5% Tween20 * incubate in primary antibody (2 h at 16oC or overnight) * rinse PBS containing 0.5% Tween 20 * incubate with secondary antibody (2h at 30oC) *** typically we are now using ALEXA-conjugated secondary antibodies from Molecular Probes (http://www.probes.com/lit/feature/alexa/). * wash in Tween PBS * mount in airvol + propyl gallate | |
| Troubleshooting | |
| Reference | |
| Klymkowsky Lab (http://spot.colorado.edu/~klym/Home.html) | |
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