Sp15 100

After collection of the tissue samples, skin explants were fixed in 10% neutral buffered formalin for 48 h at 4 °C, then dehydrated using increasing alcohol gradients, followed by immersion in xylene and paraffin embedding.
For H&E observation, 4 µm thick sections were deparaffinized in xylene and then rehydrated in decreasing alcohol gradients and then stained with Mayer’s hematoxylin solution for 10 min (26043-06, Electron Microscopy Sciences). After rinsing the section in tap water for 15 min, sections were stained with aqueous Eosin Y solution (786-1072, Biosciences) for 3 min and then dehydrated in increasing alcohol solutions, terminating with xylene step. Following, the sections were mounted onto glass slides using a toluene-based solution (SP15-100, Fisher Chemical) and images were taken with (EVOS FL Auto, Life Technologies)^{43 (link)}.

Tissue rings were fixed with 4% PFA overnight at 4°C and dehydrated with 30% sucrose solution (Fisher Scientific, S5-500) for 3 hours. Next, tissue rings were embedded with optimal cutting temperature compound (Fisher Scientific, 50363579), frozen overnight at −80°C, and then sectioned to a thickness of 10 μm with Cryostat (Sakura, 6203) in McDonald lab at Syracuse University. H&E staining was performed with the standard protocol. Tissue rings were rehydrated by alcohol with gradient concentration and counterstained with hematoxylin (Leica, 3801560) and eosin (Leica, 3801600). After dehydration with alcohol, tissue rings were cleared in xylene (Sigma-Aldrich, XX0060-4) and mounted with Permount (Fisher Scientific, SP15-100) on glass slides. Images were taken with the Leica ICC50 microscope.

Paraffin sections were cut along the superior-inferior plane through optic nerve and stained with hematoxylin (MHS16, Sigma, Burlington, MA, USA) and eosin (HT110116, Sigma, Burlington, MA, USA). The slides were mounted using permount mounting medium (SP15100, Fisher Scientific, Waltham, MA, USA), and light microscopic images presented in the figures were captured using 40X objective from the inferior side, 500 μm away from the optic nerve using a Zeiss Axioskop 50 (Carl Zeiss, White Plains, NY. USA). For quantifications, cells in the outer nuclear cells layer were counted in 100 μm windows at intervals of 200 μm across the superior-inferior plane using ImageJ.

Matching sections were taken from both PFA and glyoxal fixed brain samples into glass slides and sections were completely covered with hematoxylin for three minutes followed by rinsing in two changes in distilled water, each for 15 s, dipped in 100% ethanol for 10 s, drained off the excess ethanol and then eosin Y solution was added to the slides and incubated for 30 s. Then, sections were dehydrated using ascending grades of ethanol from 70% to 100%, cleared in xylene and mounted with permount mounting medium (SP15-100 Fisher Scientific, USA).

Briefly, formalin-fixed paraffin-embebbed tissue sections were deparaffinised, rehydrated and subjected to an antigen retrieval step, blocked with 10% Normal Serum (PK-6200, Vector Laboratories, Burlingame, USA) in DAKO diluent (S3022, DAKO, Glostrup, Denmark), washed and incubated overnight at 4 °C with 1:300 of anti-MOXD1 (bs-17733R, Bioss, Massachusetts, USA) antibody. Specific staining was detected with secondary biotinylated horse anti-rabbit antibody (PK-6200, Vector Laboratories, Burlingame, USA) followed by Vectorstain ABC Elite reagent and DAB peroxidase substrate (SK-4105, Vector laboratories, Burlingame, USA) and counterstained with haematoxylin. Slides were mounted with permount mounting medium (SP15-100, Fisher Scientific, Pittsburgh, PA, USA) and analyzed in a light microscopy (Nikon Eclipse 55i, Melville, NY, USA).