FixationwithGlutaraldehyde

vol(for500ml)ofstockmg
1.137mMNaCl34.25ml2M
5mMKCl0.83ml3M
1.1mMNa2HPO45.5ml100mM
0.4mMKH2PO42ml100mM
2mMMgCl210ml100mM
2mMEGTA10ml100mM
5mMPIPES25ml100mM
5.5mMglucose495
pH6.1(Cytoskeletonbuffer),warm.
2.Glutaraldehyde,storeintightly-sealedcontainersat4oC.
3.Cytoskeletonbufferwith0.3%(w/v)Triton-X100,0.5%glutaraldehyde,30ml.TritonX-100shouldbepipetedslowlywithap-200pipetmanandatrimmedtip.Wipetheoutsidesurfaceofthepipettipbeforeaddingtothebuffer.Pumpupanddownseveraltimestorinseouttheinsidesurface.Mixinafixativebottle,captightly,andwarminawaterbath.
4.Cytoskeletonbufferwith1%glutaraldehyde,30ml.Mixinafixativebottle,captightly,andwarminawaterbath.
5.Cytoskeletonbufferwith0.5mg/mlNaBH4,30ml.Preparefreshbeforeuseinafixationbox.BubblesshouldbevisIBLe.StoreNaBH4desiccatedinatightly-sealedcontainer.
6.Phosphatebufferedsaline,100ml,warm.
7.Fixationboxes/dished.
1.Rinsecoverslip2xwithwarmphosphatebufferedsaline.
2.Fix1mininbuffer3.Agitateperiodically.
3.Rinse2xwithcytoskeletonbuffer.
4.Fix15mininbuffer4.Agitateperiodically.
5.Rinse2xwithcytoskeletonbuffer.
6.Treat5mininNaBH4.Agitateperiodically.
7.Rinse2xwithcytoskeletonbuffer.