|Michael Klymkowsky ( klym@spot.Colorado.EDU )|
|University of Colorado, Boulder , United States|
|Protocol ID / PID|
|Tissue sectioning: Cryostat sectioning method|
|(ala Fagotto & Gumbiner, 1994)|
| MEMPfa: |
0.1M MOPS -- pH 7.4
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).
80% ethanol : 20% DMSO (can be stored at r.t. forever).
1 part 30% hydrogen peroxide & 2 parts
0.1 M maleic acid
0.15 M NaCl
Adjust to pH 7.5 with NaOH
Dilute blocking solution into MAB
| 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
| Klymkowsky Lab|
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