Tissue sectioning: Cryostat sectioning method
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| Overview | |
| (ala Fagotto & Gumbiner, 1994) | |
| Material | |
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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 |
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| Procedure | |
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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~Kfont_color~L~4blue~4~Mhttp~I~H~Hwww.probes.com~Hlit~Hfeature~Halexa~H~K~Hfont~M_~B.~Kbr_~H~M~2~1~0~0~0~0~Kbr_~H~M~2~1~0~0~0~undefined wash in Tween PBS ~undefined mount in airvol + propyl gallate |
