This is a cached page for the URL (http://iprotocol.mit.edu/protocol/197.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Tissue sectioning: Cryostat sectioning method
protocol book |  logoff |  modify profile |  post protocol |  edit protocol |  add this to protocol book  

 
 
  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 -17C 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)  
 
 Back to the Top