Methanol

Kidney specimens were fixed in 10% neutral buffered formalin (Biosesang) for 24 hours, and paraffin blocks were made according to the previously published standard methods [22 (link)]. Paraffin blocks were then cut into 4-µm sections, and paraffin-embedded tissues were de-paraffinized by changes in xylene and rehydrated in decreasing concentrations of ethanol (100%, 95%, 80%, and 70%). The sections were then stained with hematoxylin and eosin (Gill III, Merck) using an automatic strainer (Leica). For the immunohistochemistry staining of cleaved caspase-3, paraffin block sections were immersed in methanol (Duksan) with 0.3% H₂O₂ for 10 minutes at room temperature to quench endogenous peroxidase activity. Subsequently, they were washed twice with Tris-buffered saline and blocked with 5% bovine serum albumin in phosphate-buffered saline. After adding the cleaved caspase-3 antibody (1:1000 dilution, #9664, Cell Signaling Technologies) as the primary antibody for 1 hour at room temperature, they were washed twice with Tris-buffered saline and supplied with biotinylated goat-anti-rabbit IgG-HRP (1:10000 dilution, #K4003 from DAKO, Glostrup) as the secondary antibody. The stained slides were observed with fluorescent microscopy (IX71/DP71, Olympus).

Three weeks after cell transplantation (day 28), the mice were anesthetized with Zoletil (0.012 ml/20 g body weight; Virbac, St. Louis, USA) and Rompun (0.008 ml/20 g body weight; Bayer Korea, Seoul, Korea) and blood (1 ml) was collected by cardiac puncture into heparin-coated 1-ml syringes (JWphama, Seoul, Korea). Blood cells were counted using a Neubauer chamber and blood smear slides were prepared for staining. Peripheral blood smears were air-dried and dipped in methanol (Duksan, Ansan-si, Korea) three times for fixation, followed by Diff-Quik staining (Sysmex Corporation, Kobe, Japan). The femurs of each mouse were isolated following cervical dislocation, and immediately fixed with 4% paraformaldehyde (Sigma-Aldrich) and decalcified for embedding in paraffin (Korea Animal Medical Science Institute, Guri-si, Korea). BM sections (4 µm) were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) and images of the tissue sections were captured using a BX-50 microscope and a DP-71 digital camera and imaging system (DPController 3.2.276.2) (all from Olympus Corp., Tokyo, Japan). Histological analysis was conducted using Image J software version 1.49 (National Institutes of Health, Bethesda, MD, USA) by selecting adipocytes in the BM using a color threshold. Other cells (erythroid and myeloid cells) were selected for BM area measurement.

PCL (average molecular weight: 80,000; Sigma-Aldrich Co., St Louis, MO, USA) was dissolved at 1.5 g/mL in a solvent mixture of 9:1 (v/v) chloroform (Junsei, Tokyo, Japan), and methanol (Duksan, Gyeonggi-do, South Korea). The electrospinning apparatus consisted of a syringe pump (KD Scientific, Holliston, MA, USA), a 10-mL syringe (Becton Dickinson, Franklin Lakes, NY, USA), a needle tip (23G; Becton Dickinson), a high-voltage power supply (KSC Inc., Seoul, South Korea), an earth grounder, and a collector (Figure 1A). To produce the highly porous 3D electrospun scaffold, a novel collector with a void (0.5 cm diameter and 0.95 cm depth) was designed on a hexagonal polystyrene weighing dish (0.44×0.95 cm; Eagle Inc., Hodgenville, KY, USA) wrapped with an aluminum foil (16 μm thick).24 A traditional collector with the same geometry as the novel collector, but without the void space, was also wrapped in aluminum foil for producing the 2D scaffold. Scaffolds (3D and 2D) were fabricated at the rate of 0.2 mL/h at 10 kV. The distance between the collector and needle tip was 8 cm, and the deposited volume of the solution was 0.2 mL.