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Sections were rinsed in PB, cryoprotected in 30% sucrose in PB overnight, and frozen in liquid nitrogen 3 times. After extensive washes in PB, sections were treated with 1% sodium borohydride in PB for 5 or 10 min (10 min in cases when both the perfusing and the immersion fixative contained glutaraldehyde). Then, sections were washed in PB and 0.05 M TBS (pH 7.4) and blocked in 1% HSA (Sigma-Aldrich) in TBS. Then, sections of virus-injected mice were incubated in a solution of chicken anti-eGFP (1:2,000, Thermo Fisher Scientific, A10262) and mouse anti-gephyrin (1:100, Synaptic Systems, 147021) primary antibodies diluted in TBS containing 0.05% sodium azide for 2 days. After repeated washes in TBS, the sections were incubated in a blocking solution (Gel-BS) containing 0.2% cold water fish skin gelatin and 0.5% HSA in TBS for 1 h. Next, sections were incubated in a mixture of Nanogold-Fab’ goat anti-mouse IgG (1:100, Nanoprobes, 2002) and goat anti-chicken biotinylated secondary antibodies (1:200, Vector Laboratories, BA-9010) in Gel-BS overnight at 4°C.
Sections of BDA-injected mice prepared for electron microscopic analysis were treated similarly, except that anti-eGFP primary and biotinylated secondary antibodies were not added to the solutions. After extensive washes in TBS, the sections were treated with 2% glutaraldehyde in 0.1 M PB for 15 min to fix the gold particles into the tissue. After TBS washes, sections were incubated in Elite ABC (1:300, in TBS) overnight or for 2 days. To enlarge immunogold particles, sections were incubated in silver enhancement solution (SE-EM; Aurion) for 30 to 40 min at room temperature.
In the case of mice used for Neurolucida drawing of whole dendritic trees (n = 6), sections were treated similarly except that they were not incubated in anti-gephyrin primary- and gold-conjugated secondary antibodies, GEL-BS-, glutaraldehyde-, and SE-EM solutions. The immunoperoxidase reaction was developed using 3,3-diaminobenzidine (DAB; Sigma-Aldrich) (all virus-injected mice and n = 5 BDA-injected mice) or ammonium nickel sulphate-intensified DAB (n = 1 BDA-injected mouse used for Neurolucida drawing of whole dendritic trees) as chromogen (Fig 1).

For electron microscopy, MSC were plated in 8 well Lab-Tek Permanox slide chambers and cultured for 48 h and then NALM6 cells were added into MSC-cultured chamber and cultured for 48 h. Nonadh NALM6 cells were washed out by PBS. Adherent NALM6 with MSC were fixed by EM fixative composed of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1M phosphate buffer (PB) for 10 min at room temperature. After removal of fixative and immediate washing with PB, co-cultured sample was quenched with 0.1% sodium borohydride in PB. Integrin beta 1 is visualized by labeling with anti-Integrin beta 1 antibody (P5D2) from Abcam, an ultrasmall gold conjugated goat anti-mouse antibody (Aurion) and silver enhancement (Aurion R-Gent SE-EM). Following labeling and silver enhancement, samples were osmicated, contrasted en bloc with uranyl acetate, dehydrated and infiltrated in EmBed-812 resin (Electron Microscopy Sciences). Fully infiltrated samples were polymerized at 60°C for 48 h. Samples were sectioned at 70nm on a Leica UC-7 ultramicrotome and imaged in a ThermoFisher Scientific TF-20 operating at 80kV equipped with an AMT NanoSprint15 MkII imaging system.