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        Tissue sectioning: Cryostat sectioning method

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        1127

         

        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 )  

         

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