Agarose is permeable for very large molecules (several MDa). It was used for immune electrophoresis before blotting became available. For your application, however, you'd probably have to make frozen sections from the agarose blocks. Fish skin gelatin is an ugly looking liquid, which reminds me of used motor oil. It does not solidify like mammalian gelatin does. Very good as carrier protein for washing buffers though, I got really clean blots in situations where milk powder did not work. Whether your membranes adhere to poly-Lys slides you have to try.

Hi

I have quite a bit of experience with IEM on isolated nuclear envelope (NE) and nuclei fractions from budding yeast. You can find a description of the method in the attached paper.

In brief, cut out 4 wells from a 96-wells microtiter plate and incubate with 2.5% glutaraldehyde in water for 30 min at 25C. Remove the fixative solution and wash wells out briefly under a gentle flow of water. Add 100 micro liters of 1% poly-L-lysine in water and continue the incubation for 30 min at 25C. With this pre-treatment poly-L-lysisne is crosslinked to the plastic walls of the wells and this will in turn increase their capacity to retain immobilized membranes. Also in my experience this reduces their tendency to clump up. Dilute membrane fraction gradually to 1.0M sucrose using your buffer of choice before harvesting them by centrifugation at ~24'000 g for 2 hr (N.B. this will depend of course on the sedimentation characteristics of your membrane fraction) at 4C, to the bottom of the pre-treated microtiter wells. After harvesting, wash the membrane fractions in your buffer of choice and then fix them with a formaldehyde solution diluted in the same buffer (N.B. Fixation conditions will vary depending on the antigen you want to stain by IEM and they will have to be tested. In my case I fixed in 4% formaldehyde for 5-10 min at 25C). Membranes are subsequently washed first in the original buffer and then in the blocking solution that will be used for IEM (N.B in my case the IEM buffer consisted of 0.5% BSA in 0.5x PBS-K, pH 7.0 containing 1 mM MgCl2, 10 microM CaCl2 and 10 microM ZnCl2, and protease inhibitors). After that you can proceed with primary and secondary antibody binding, glutaraldehyde fixation, and osmium treatment before preparation for transmission EM.

Hope this helps.

C

Article Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior