Alcian blu

Skeletal muscle or myocardium fragments from Lmna^+/+ or Lmna^G609G/G609Gmice were frozen in melting isopentane and stored in liquid nitrogen. Unfixed cryosections were subjected to immunofluorescence staining as detailed above.
Samples of aortic arch promptly after necropsy were fixed with formalin and embedded in paraffin. Histology was based on hematoxylin–eosin, PAS stain (Bio Optica, Milan, Italy; P.A.S. Hotchkiss – MC Manus, 04‐130802) and Alcian stains at pH 2,5 and 1 (Bio Optica; Alcian Blu, 04‐160802). Reduction of cellularity in aorta middle coat was graded as described (Zaghini et al., 2020) and reported in Table S1. For aorta examination, three photographs per sample were acquired with a DFK 33UX264 camera coupled with a Leica TM DMLB microscope (TIFF format 2448 × 2048, Obj 20×).
The area was manually delineated (lowest selected area = 40,000 µm²), and leiomyocytes were manually counted with ImageJ software (https://imagej.nih.gov/ij/index.html/). Finally, cellularity was assessed and expressed as the number of leiomyocytes/10,000 µm².

Histological sections (5 μm) of control and 0.1% SDS-treated lenticules, fixed in formalin and embedded in paraffin (n = 6), were stained with Hematoxylin and Eosin (H&E), Alcian Blu (Bio-Optica, cat^# 04-160802) and Periodic Acid-Schiff (PAS; Bio-Optica, cat^#04-130802) kits. The sections were photographed under light microscopy using 10X and 20X objectives. Lenticules thickness (5 standard position) and staining intensity of PAS and Alcian Blue were measured by using ImageJ software (NIH, United States ImageJ software, public domain available at: http://rsb.info.nih.gov/nih-image/).