Tecnai g2 twin electron microscope

To prepare for EM visualization of the β₂AR-aptamer complexes, affinity-purification using biotin/NeutrAvidin system was employed, as described above, for pull-down assays. An anti-FLAG Fab was developed to specifically label the FLAG-tagged β₂AR at its extracellular N-terminus. This Fab was derived from a monoclonal mouse anti-FLAG M1 IgG that recognizes the FLAG-epitope was produced using hybridoma technology for antibody production^{43 (link)}. The anti-FLAG Fab was isolated by digestion of the monoclonal mouse anti-FLAG M1 IgG on an immobilized-papain protease resin and followed by purification on a Protein-A column (Pierce)^{43 (link)}. The β₂AR and aptamer complexes (assembled as 10 µM and 20 µM in 125 µL volume, respectively) were formed in a buffer composed of 20 mM HEPES, pH 7, 25 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 2mM CaCl₂, 0.01% MNG, 0.001% CHS and 10 µL ligand. β₂AR-aptamer complexes were eluted in a buffer that has 4 mM biotin and then prepared for EM using conventional negative-staining protocols as described previously^{44 (link)}. Specimens were imaged at RT with a FEI Tecnai G² Twin electron microscope operated at 120 kV using low-dose procedures. Images were recorded at a magnification of ×65,200 and a defocus value of ~1.5 µm on an Eagle 2K CCD camera.

A2EN^Cas9 and A2EN EPS8 KO cells were cultured in collagen-coated six well plates for four days. Cells were rinsed twice with Hank’s Buffered Saline Solution and fixed with buffer containing 0.05% malachite green, 2.5% glutaraldehyde, 100 mM sodium cacodylate (pH 6.7) for two hours at 25C. Cells were washed four times for five min with 100 mM sodium cacodylate and stained with 0.8% K3Fe(CN)6 with 1% osmium tetroxide. The cells were rinsed four times for 5 min with 100 mM sodium cacodylate, stained with 1% tannic acid for 20 min, and washed once for 5 min with 100 mM sodium cacodylate and twice for 5 min with molecular grade distilled water. The cells were stained with 0.5% uranyl acetate overnight at 4C, washed five times for 5 min with molecular grade distilled water, dehydrated with a series of ethanol incubations, and embedded in resin and vertically cut. Ultrathin sections were post-stained with uranyl acetate and lead citrate. Images were acquired on a FEI Tecnai G² Twin electron microscope at 6500x magnification.