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Cells were next stained with Alexa Fluor 488 conjugated secondary antibody (Invitrogen) in blocking buffer, the nuclei stained with Hoechst 33342 (1 µg/ml in PBS; Invitrogen) and washed (2×5 min) and stored in PBS.

Tissue sections from saline and S. pneumoniae-infected mice were next stained for fibrin ogen) by immunohistochemistry (red stain, Fig. 4b).

Tissue sections from S. pneumoniae-infected WT and PAI-1−/− mice were next stained for fibrin (ogen) by IHC (Fig. 8c).

The cells were next stained with 100 μL 0.4% SRB (Sigma, USA) at 37 °C for 30 min, and subsequently washed five times with 1% acetic acid to remove unbound stain.

Cells were next stained for one second with 3.6 mM DAPI in PBS, followed by three rinses with PBS.

Paraplast transverse sections, 6µm in thickness, being cut with a microtome were next stained with hematoxylin and eosin (H&E) for general morphological observation, and with Masson's trichrome (MT) to visualize the collagen fibers.

The cells were next fixed, permeabilized and stained with a secondary antibody to determine the sub-cellular localization of the furin-antibody complex.

The cells were next washed with PBS before staining with the DNA dyes as described earlier.

Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole.

Infiltrating macrophages were next analyzed by ED2 staining, as described previously.

For immunofluorescence staining, cells were next treated with 0.5% Triton X-100 in PBS for 15 minutes and blocked with 10% BSA in PBS for 1 hour followed by incubation with anti-p65 antibody at 4°C overnight.