Mouse monoclonal anti vimentin antibody

To detect macroglia cells, retinae were stained with antibodies against glial fibrillary acidic protein (GFAP, astroglia), vimentin and glutamine synthetase (Müller glia) 14 days after immunization (n = 5/group). In addition, optic nerves were stained with an antibody against GFAP at day 14.
The retinal cross‐sections were incubated with 10% donkey and/or goat serum in 0.1% Triton‐X in PBS for 30–60 min. A mouse monoclonal anti‐GFAP, directly labelled with Alexa Fluor 488 (1:1.200; Millipore, Darmstadt, Germany), a mouse monoclonal anti‐vimentin antibody (1:500; Sigma‐Aldrich) and a mouse monoclonal anti‐glutamine synthetase antibody (1:200; Invitrogen) were applied overnight. The corresponding secondary antibodies goat antimouse Alexa Fluor 555 (1:400; Invitrogen) and goat antimouse Alexa Fluor 488 (1:500; Invitrogen) were added the next day for 60 min. Optic nerve sections were incubated with a chicken monoclonal GFAP antibody (1:500; Millipore) overnight. The secondary antibody, donkey anti‐chicken Cy3 (1:500; Millipore), was applied the next day for 60 min. Cell nuclei were visualized on retinal and optic nerve sections with DAPI. The photographs were taken using a fluorescence microscope (Axio Imager M1; Zeiss). Macroglia stainings were analyzed using an ImageJ macro, as describes above.

F-actin and vimentin staining was performed according to previously described protocol [59 (link)]. In short, A549 cells grown on sterile glass coverslips were fixed with 4% paraformaldehyde (Serva, Heidelberg, Germany) and permeabilized with 0.25% Triton X-100. F-actin was labeled with phalloidin conjugated to tetramethylrhodamine isothiocyanate (TRITC diluted 1:5 in PBS, 20 min; Sigma-Aldrich, St. Louis, MO, USA). Non-specific background was blocked with 1% bovine serum albumin (BSA). Mouse monoclonal anti-vimentin antibody (diluted 1:50 in BSA-PBS, 60 min; Sigma-Aldrich, St. Louis, MO, USA) was used as a primary antibody to label vimentin (diluted 1:50 in BSA-PBS, 60 min; Sigma-Aldrich, St. Louis, MO, USA), followed by the incubation with a secondary goat anti-mouse antibody conjugated to Alexa Fluor 488 (diluted 1:100 in PBS, 60 min; Sigma-Aldrich, St. Louis, MO, USA). Cell nuclei (DNA) were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA). All of the incubations were done at ambient temperature, whereas fluorescent staining was performed in the dark. Finally, slides were mounted in Aqua-Poly/Mount (Polysciences, Warrington, PA, USA) and examined using a Nikon Eclipse E800 fluorescence microscope and NIS-Elements 4.0 software (both from Nikon, Tokyo, Japan).