Journal: Parasites & Vectors

Article Title: Role of gamma-giardin in ventral disc formation of Giardia lamblia

doi: 10.1186/s13071-019-3478-8

Figure Lengend Snippet: Effect of morpholino-mediated knockdown of Glγ-giardin on Giardia cell division. a Giardia trophozoites were collected 48 h after electroporation with control morpholino (control mph; Lane 1), or anti-Glγ-giardin morpholino (anti-Glγ-giardin mph; Lane 2). Extracts of these cells were reacted with anti-Glγ-giardin or anti-GlPDI1 antibodies. Abundance of each immunoreactive protein was quantified by densitometry and normalized to that of Giardia treated with control morpholino. b Expression of Glγ-giardin was monitored by IFAs. Cells were reacted overnight with rat anti-Glγ-giardin polyclonal antibodies (1:100) then treated with Alexafluor 555-conjugated anti-rat IgG (1:100). Slides were mounted with VECTASHIELD anti-fade mounting medium with DAPI then observed with an Axiovert 200 fluorescent microscope. c Effect of anti-Glγ-giardin morpholino on cytokinesis of Giardia cells. Cells were stained with 10% Giemsa solution and observed with a light microscope to count numbers of cells in interphase (gray columns), mitosis (closed columns) and cytokinesis (open columns). d Cell number was determined by counting a least 500 cells per each condition. Cells were attached on coverslips and mounted in anti-fade mounting medium with DAPI. To determine numbers of cells with four or two nuclei, more than 300 cells per condition were counted. Scale-bars : b , c , d , 2 µm

Article Snippet: Following three 5-min washes with PBS, the cells were incubated with Alexafluor 488-conjugated anti-mouse IgG (1:100; Molecular Probes, Grand Island, NY, USA) and Alexafluor 555-conjugated anti-rat IgG (1:100; Molecular Probes) at 37 °C for 1 h. Slides were mounted with VECTASHIELD anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).

Techniques: Electroporation, Expressing, Microscopy, Staining, Light Microscopy