4 6 diamidino 2 phenylindole dihydrochloride dapi

Lumogallion staining was performed on formalin-fixed hippocampal tissue using a recently validated method to identify the presence of Al in tissues^{44 (link),45} . Briefly, re-hydrated tissues sections were immediately placed into either 1 mM lumogallion (TCI Europe N.V. Belgium) buffered in 50 mM PIPES, pH 7.4 or the PIPES-buffer alone for auto-fluorescence analyses for 45 minutes. Slides were carefully washed 6 times with PIPES-buffer and rinsed in ultrapure water for 30 seconds. Sections were then incubated with DAPI to identify the cell nucleus (4′,6-Diamidino-2-phenylindole dihydrochloride - DAPI, 1:10.000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Afterwards, slides were mounted using an aqueous mounting medium and stored horizontally at 4 °C overnight prior to imaging. Sections of tissues were imaged using a Zeiss Axioskop 2 microscope (Zeiss, Jena, Germany).

The hearts were excised and fixed in 10% formalin, embedded in paraffin, and sectioned at 5-μm thickness. The picrosirius red stain and Masson’s trichrome stain were used to detect collagen content in the cardiac tissues. The positively stained areas of the left ventricular myocardium were examined by using a 100× objective and an average of five fields per mouse was calculated. The index of the positively stained area was calculated as the percentage of positively stained cells per the total area of the left ventricle.
For immunofluorescent staining, slides were incubated with primary antibodies against fibronectin (Abcam, Cambridge, UK), vimentin (Abcam, Cambridge, UK), CD11b (Biolegend, San Diego, CA, USA), monocyte chemoattractant protein-1 (MCP-1) (Abcam) at 4 °C overnight, and then with a fluorescent dye-conjugated secondary antibody (Invitrogen). Nuclei were visualized by staining with 4′6-diamidino-2-phenylindole dihydrochloride (DAPI) (Santa Cruz Biotechnology, Dallas, TX, USA). The positively stained cells of the left ventricular myocardium were examined by using a 20× objective and an average of five fields per mouse was calculated. The index of positively stained area was calculated as the percentage of positive cells per total number cells. All of the images were captured using immunofluorescent microscope (Olympus BX53, Tokyo, Japan).

Mouse BMMs were plated on glass coverslips in the presence or absence of 5 μM OCLI-023. After 4 days in culture, the cells were fixed with 4% paraformaldehyde, treated with 0.1% Triton X-100, and stained with rhodamine-conjugated phalloidin (Cytoskeleton, Denver, CO, USA) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Santa Cruz Biotechnology, Santa Cruz, CA, USA) to visualize F-actin and nuclei, respectively. Fluorescent images were obtained using a BX51 fluorescence microscope (Olympus, Tokyo, Japan).

Spheroids were fixed overnight in 4% paraformaldehyde and permeabilized for 5 minutes with 0.1% Triton X-100 in PBS. Samples were then blocked for 1 hour using 0.01% Tween-20, 10% goat serum and 1% bovine serum albumin (BSA) in PBS. Afterwards, samples were incubated with HIF1 alpha primary antibody (Abcam; ab179483; rabbit monoclonal; 1:50) or MMP9 primary antibody (Abcam; ab76003; rabbit monoclonal; 1:250) overnight at 4 degrees, and then incubated with Goat Anti-Rabbit IgG H&L Alexa Fluor® 488 secondary antibody (Abcam; ab150077; goat polyclonal; 2 µg/mL) for 2 hours, as necessary. For F-actin staining, samples were incubated with CytoPainter Phalloidin-iFluor 555 reagent (Abcam; ab176756; 1:1000) for 2 hours. Samples were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Santa Cruz; 1.5 µg/mL). All blocking and incubations were performed at room temperature, unless otherwise stated. Spheroids were visualized using an Eclipse Ti-E Inverted Confocal Microscope (Nikon). Z-stack images were taken in 10 µm steps to capture all layers and compiled as a z-projection image. Images were analyzed using NIS Elements software (Nikon).