Axio image d2 microscope

Tissue samples were fixed in 4% paraformaldehyde overnight, dehydrated using a graded alcohol series, then embedded in paraffin, and processed to obtain tissue sections for HE staining. Images were obtained using an Axio Image D2 microscope (Zeiss) and photographed using Axio Image D2 digital camera.

Tissue sections were deparaffinized, rehydrated, and washed three times with Tris-buffered saline (Sigma). Sections were incubated with H₂O₂ for 30 min and washed with distilled water to avoid nonspecific background staining. Adipose tissue samples were stained with anti-CD34 (Abcam) and guinea pig anti‐mouse perilipin (Progen) antibodies following the manufacturers’ instructions. After washing, the samples were incubated with goat anti-rabbit Alexa Fluor® 488 IgG (Abcam) and goat anti-guinea pig Alexa Fluor® 647 IgG (Abcam). Nuclei were stained with DAPI (Sigma, USA).
For migrasomes detection, tissue samples were stained with anti-TSPAN4 (Biossusa), anti-TSPAN7 (Biorbyt) antibodies following the manufacturers’ instructions and then with goat anti-rabbit Alexa Fluor® 488 IgG (Abcam) and goat anti-rabbit Alexa Fluor® 647 IgG (Abcam). Nuclei were stained with DAPI (Sigma, USA). Furthermore, the tissue samples were stained with anti-integrin β1 (SantaCruz), and then with goat anti-mouse Alexa Fluor® 488 IgG (Abcam). Nuclei were stained with DAPI (Sigma). Images were obtained using Axio Image D2 microscope (Zeiss) and analyzed using ImageJ software.