Hematoxylin reagent

Adipocyte lipolysis was assessed using an Oil Red O stain (O0625, Sigma–Aldrich, St. Louis, MO, USA) as an indicator of intracellular lipid accumulation. After the irradiation-induced lipolysis, cells were washed twice with PBS, fixed with 10% formalin in PBS for 1 h, and then washed with 60% isopropanol, before being allowed to dry completely. Adipocytes were stained with 0.2% Oil Red O reagent for 10 min at room temperature, and washed with H₂O four times. Each sample was eluted with 100% isopropanol for 10 min and absorbance was measured at 505 nm using a spectrophotometer. To visualize the nucleus, adipocytes were counterstained with hematoxylin reagent (H3136, Sigma–Aldrich, St. Louis, MO, USA) for 2 min and washed twice with H₂O. The level of adipocyte differentiation was observed using an inverted phase-microscope.

The process of staining was performed according to Abumadighem et al. (2018) [2 (link)]. The cut of the tissue on the slides was deparaffinized in K-clear solution (Kaltek), rehydrated in ethanol (100%, 70%, and 50%), and then rehydrated with deionized water. Next, the slides were incubated in a hematoxylin reagent (sigma) (1:5 phosphate-buffered saline–PBS) and then rinsed with tap water. This was followed by a short dip in eosin (Kaltek, Padova, Italy) before then being rinsed with distilled water. Slides were then dehydrated with ascending ethanol concentrations of 50%, 70%, and 100%, followed by the use of K-clear. Finally, the slides were dried and covered by a coverslip using Permount (Thermo Fisher Scientific, Denver, CO, USA)

Formalin-fixed tissue samples were processed into paraffin blocks and sectioned by the TRI Histology Core Service. After de-waxing in xylene and re-hydrating through ethanol gradients (100%, 95%, 90% 70%, and dH₂O), paraffin sections were placed in Hematoxylin reagent (Gill’s formula, No.2, Sigma-Aldrich) for 30 s and then washed under running tap water for 5 min. Slides were then placed in Eosin Y reagent (Sigma-Aldrich) for 30 s. After Eosin staining, slides were dehydrated in 90%, 100% ethanol and xylene, followed by mounting with Pertex® mounting medium (Medite, Burgdorf, Germany) and examination under a bright field microscope.