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Tissue sectioning: Cryostat sectioning method
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  Author Information
  Primary Author
  Michael Klymkowsky ( klym@spot.Colorado.EDU )
  University of Colorado, Boulder , United States
  Protocol Information
  Protocol ID / PID
  Cell Biology
  Tissue sectioning: Cryostat sectioning method  
  (ala Fagotto & Gumbiner, 1994)  

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).

eDents bleach:
1 part 30% hydrogen peroxide & 2 parts

0.1 M maleic acid
0.15 M NaCl
Adjust to pH 7.5 with NaOH

Blocking Buffer:
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 -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 (

* wash in Tween PBS

* mount in airvol + propyl gallate
  Klymkowsky Lab
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