Anti gpc1pipa528055

Fixed specimens at an optimal concentration were placed onto a 400 mesh
carbon/formvar coated grids and allowed to absorb to the formvar for a minimum
of 1 minute. For immunogold staining the grids were placed into a blocking
buffer for a block/permeabilization step for 1 hr. Without rinsing, the grids
were immediately placed into the primary antibody at the appropriate dilution
overnight at 4°C (1:300 anti-CD9 ab92726, Abcam and anti-GPC1
PIPA528055, Thermo Scientific). As controls, some of the grids were not exposed
to the primary antibody. The following day, all the grids were rinsed with PBS
then floated on drops of the appropriate secondary antibody attached with 10 nm
gold particles (AURION, Hatfield, PA) for 2 hrs at room temperature. Grids were
rinsed with PBS and were placed in 2.5% Glutaraldehyde in 0.1M Phosphate
buffer for 15 min. After rinsing in PBS and distilled water the grids were
allowed to dry and stained for contrast using uranyl acetate. The samples were
viewed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro,
OR) and images were taken with an AMT CCD Camera (Advanced Microscopy
Techniques, Danvers, MA).

Cells were lysed in RIPA buffer containing 5
μg.ml⁻¹ leupeptin, 1
μg.ml⁻¹ pepstatin and 1mM phenylmethylsulphonyl
fluoride. Exosomes were lysed in 8M Urea/2.5% SDS containing 5
μg.ml⁻¹ leupeptin, 1
μg.ml⁻¹ pepstatin and 1mM phenylmethylsulphonyl
fluoride. Sample loading was normalized according to Bradford relative protein
quantification and proteins separated following an electrophoretic gradient
across polyacrylamide gels. Wet electrophoretic transfer was used to transfer
the proteins in the gel onto PVDF membranes (ImmobilonP). The protein blot was
blocked for 1hr at room temperature with 5% non-fat dry milk in
PBS/0,05% Tween and incubated overnight at 4°C with the
following primary antibodies: 1:300 anti-GPC1, PIPA528055 (Thermo-Scientific);
1:300 anti-β-Actin A3854 (Sigma-Aldrich); 1:300 anti-CD81 sc-166029
(Santa-Cruz); 1:300 anti-Flottilin1 sc-25506 (Santa-Cruz). Afterwards, HRP
conjugated secondary antibodies were incubated for 1 hr at room temperature.
Washes after antibody incubations were done on an orbital shaker, four times at
10 min intervals, with 1X PBS 0.05% Tween20. Blots were developed with
chemiluminescent reagents from Pierce.