Tissue sectioning: Cryostat sectioning method

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

~undefined 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.

~undefined Embed in 15% sucrose/7.5% gelatin, chill to 4oC (you can hold the samples here for a few days).

~undefined 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).

~undefined 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.

~undefined Place the block into the cyrostat and allow it to equilibrate to the cutting temperature (i.e. -17 oC)

~undefined Prepare 12-14 mm thick sections using a cryostat at -17� 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).

~undefined Warm slides and allow them to dry at room temperature

~undefined extract for 2 min. in 100% acetone.

~undefined rehydrate in PBS

~undefined block with TNB blocking buffer (from tyramide kit) for 30 min at r.t. or just incubate in PBS + 0.5% Tween20

~undefined incubate in primary antibody (2 h at 16oC or overnight)

~undefined rinse PBS containing 0.5% Tween 20

~undefined incubate with secondary antibody (2h at 30oC)
~undefined*_typically_we_are_now_using_ALEXA~Fconjugated_secondary_antibodies_from_Molecular_Probes_~A~K~Hfont~M_~Kfont_color~L~4blue~4~Mhttp~I~H~Hwww.probes.com~Hlit~Hfeature~Halexa~H~K~Hfont~M__~Kfont~M~B.~Kbr_~H~M~2~1~0~Kbr_~H~M~2~1~undefined wash in Tween PBS

~undefined mount in airvol + propyl gallate
      Troubleshooting         Reference   Klymkowsky Lab
( http://spot.colorado.edu/~klym/Home.html )