Axiovert m200

To avoid oxidation of La protein during the fixation procedure, the cells were fixed with methanol of analytical grade delivered and stored in a brown glass bottle (Merck KGaA, Darmstadt, Germany) [33 (link)]. Cells were fixed for at least 1 h or overnight. Prior to the staining, the cells were rehydrated with PBS (5 min) and stained with the respective anti-La mAb (e.g., [34 (link),35 (link),104 (link),105 (link)]). For the oxidation of fixed cells, cells were washed with PBS containing H₂O₂ (160 mM). For reduction of fixed cells, cells were washed with PBS containing ß-mercaptoethanol (5 mM). Cells were documented using an Axiovert M200 epifluorescence microscope and the software AxioVision (Carl Zeiss Microscopy GmbH, Jena, Germany). For quantitative analysis, we used a Keyence microscope and the software package BZ Analyzer II (Keyence Microscope Europe, München, Germany).

Cells were seeded in 6-well culture plates, at a density of 50,000 or 100,000 cells per well. Cells were allowed to attach overnight in regular media. After attachment, the cells were either treated with DMSO or with various concentrations of MF for 72 h. At the end of the incubation, plates were washed with PBS, and fixed with 4% PFA for 20 min, after which they were stored in PBS at 4 °C until processed for fluorescence staining as previously described. Images were taken by fluorescence microscopy with a Zeiss Axiovert M200.

Phase contrast microscopy was used to image non-treated cells, cells following exposure to treatments, and cells plated in clonogenic survival assays. Images were taken using a Zeiss Axiovert M200 inverted microscope (Carl Zeiss, Thornwood, NY). All images were taken with the objectives of 5× or 20×.

Samples of control or exposed C. elegans nematodes were immobilized with 0.2 mM levamisole hydrochloride (Vetranal, Sigma–Aldrich), mounted on glass slides and imaged by light microscopy (Olympus CK40 or Leitz Fluovert FS) or fluorescence microscopy (Zeiss Axiovert M200). Nematodes on optical 96-well plates were also directly imaged using the Zoe imager (Bio-Rad Laboratories, Hercules, CA, USA). The intensity of fluorescence was measured from digital images using the ImageJ software (Wayne Rasband, NIH, USA).

Cell morphology was examined by Hoechst 33258 and PI double staining for
live-cell imaging. After various treatments, cells were stained with Hoechst
33258 and PI solution at room temperature for 10 min. Cells were washed twice
with PBS and observed with inverted fluorescence microscope (Carl Zeiss Axiovert
M200).

Paraffin sections (3 µm) of aortic vessels from WT and α-GAL-Tg/KO mice were deparaffinized and incubated in a 10 mM citrate buffer at 95°C for 40 min for antigen retrieval. Afterward, the sections were incubated for 10 min in 0.5% Triton/TBS for permeabilization, washed in TBS, and quenched in a 0.25% Sudan black solution for 30 min on a shaker in the dark. After repeated washing, the sections were blocked in 3% bovine serum albumin (BSA) in TBS for 1 h at room temperature. The sections were then incubated with primary antibodies [anti-angiostatin (Abcam ab2904), 1:20 in 3% BSA or anti-VEGFα (Abcam, ab1316), 1:400 in 3% BSA] overnight at 4°C, washed in TBS, and incubated with secondary antibodies [donkey anti-mouse Alexa Fluor 488 (A21202 life tech) or donkey anti-rabbit Alexa Fluor 488 (21206 life tech), 1:500 in 3% BSA] for 2 h at room temperature. After repeated washing in TBS, the slides were mounted with DAPI Fluoromount G (Biozol) and fluorescence signals were descriptively analyzed using a Zeiss Axiovert M200 with ApoTome.