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Lack of staining in cells depleted of Vangl2 with a specific morpholino confirmed antibody specificity (Fig. 2D).
Lack of staining or staining <10% of tumour cells was scored as negative (Lam et al, 2008).
The specificity of our antibody was further confirmed by the lack of staining seen in our negative controls (primary antibody replaced by TBS).
Lesions with areas of lack of staining were restained at two higher antibody concentrations.
The coordinates of the points on the cellular outline were extracted manually to obtain a smooth outline for each image extending from the bottom to the top of the cell until the outline was difficult to identify due to the lack of staining in the top region.
Viability was assessed by mobility and lack of staining after challenge with trypan blue.
Viability was assessed by mobility of the parasite flagellum and lack of staining after challenge with trypan blue.
The specificity of the IF assay was supported by the lack of staining seen in the pre-treatment biopsies in any dogs (Fig. 2A, B).
Specificity of antibody labelling was demonstrated by the lack of staining after substituting proper control immunoglobulins (Rabbit primary antibody isotype control, Invitrogen) for the primary antibodies.
Indeed hMDMs maintained an intact plasma membrane up to 5 days post-phagocytosis as discerned by a lack of staining with propidium iodide (Fig. 1A), an impermeable fluorophore which can only enter cells with compromised plasma membranes.
Viable cells were distinguished by the lack of staining with propidium iodide (1 µg/ml) and considered positive for ALDH based on a control staining reaction using the enzyme inhibitor diethylaminobenzaldehyde (DEAB).
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