Ls002594

Primary murine LADC cells were obtained as previously described (41 (link)). Lung lobes were dissected out and immediately placed in 4 mL of medium A (AdMEM/F12, B27, N2, 2% FCS, 1% penicillin/streptomycin, 10 μg/mL insulin, 20 ng/mL EGF, 20 ng/mL FGF, 100 μg/mL Primocin) into GentleMACS C tubes and subjected to automated homogenization using GentleMACS dissociators at Tumor 2 mode, followed by addition of 300 U/mL collagenase IV (Worthington, LS004186) and hyaluronidase 300 U/mL (Worthington, LS002594), as well as incubation for 20 minutes at 37°C with constant shaking. Homogenized lungs were subjected to further dissociation at Tumor 3 mode, followed by centrifugation at 150g for 5 minutes at 4°C. The pellet was resuspended in medium A, and cells were plated onto Ultra-low attachment plates and placed in a humidified incubator at 37°C, 5% CO₂. Ninety-six hours later, the medium was replaced with DMEM (10% FCS, 1% penicillin/streptomycin), and cells were replated on standard tissue culture plates. All cell lines were tested as mycoplasma negative by PCR assay.

Skin tissue after different treatments were harvested, fixed in 10% neutral buffered formalin and paraffin embedded. 5 µm thick sections were cut and mounted onto slides. Slides were deparaffinized and rehydrated to TBS following standard protocol. Sections were blocked in TBS containing 10% fetal bovine serum (FBS) + 1% BSA, followed by staining with biotinylated HABP with 0.4 μl of (0.5 μg/μl stock) (EMD Millipore, 385911) HABP/100 μl + 1% BSA in PBS applied overnight. Slides were then developed with Streptavidin and DAB with Hematoxylin counterstaining and covered using Aquatex. Parallel sections that went through the whole procedure, with the only exception of HABP application, served as no staining controls, while sections that were pretreated for an hour with 1 mg/ml hyaluronidase (Worthington Biochem, LS002594) at 37 °C served as negative controls to demonstrate the specificity of the staining.
Histology slides were imaged with Leica DM 2000^® with Leica application Suite X^® version 3.0.4.16529. 40× magnification images were taken and the skin tissue was qualitatively analyzed for changes in HA staining patterns in the epidermis, dermis, and fat tissue.