🚀 go-pugleaf

 Hey Folks -- Much thanks to everyone that replied to my recent question
about immunolabeling of thick sections.  A summary of the various
responses follows:

        Pretreatment of thick cryosections (or whole mounts)of fixed tissue
        seems to often be necessary for penetration of antibodies.
        Some methods (in no special order):
                1)  Dehydrate the specimen through a graded series of
                    ethanol and then rehydrate backwards through the same
                    series.  Some include a change in xylene.
                2)  Incubate in 20mM EDTA (in H2O) 37C, 30'.
                3)  Rinse several times in DMSO.
                4)  Rinse in glycerol.
                5)  Incubate with 5% Triton X-100 (in PBS) at 4C, 1-3 days.
                6)  Several cycles of freeze/thaw.

        After pretreatment to permeabilize the specimen, typical
        quenching (eg. NH4Cl) and blocking (eg. BSA, or fish skin gelatin)
        steps, the specimen is incubated in the primary antibody for LONG
        periods of time -- often 2-3 days.  Some people recomend detergents
        during this step, others don't, but most carry out this step at room
        temp.  Incubation in seconday antibody is generally shorter --
        overnight to 24 hrs.

Thanks again to everyone who sent their methods.  I'll let them know
directly the results of my trials -- as well as anyone else who's
interested.
                        Greg Martin (gmartin@welchlink.welch.jhu.edu)
                        Dept. of Cell Biology and Anatomy
                        Johns Hopkins School of Medicine