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The differences were considered statistically significant at P?[http://assets.twoorb.com/forum/discussion/321106/the-most-up-to-date-isothipendyl-is-double-the-enjoyable#Item_1 The Most Up-To-Date isothipendyl Is Double The Enjoyable] punches), serum-free and supplemented media (DMEM, WE), and two basic assay designs (ALI and submerged) were compared over time. This revealed that 4?mm punches cultured at the ALI in supplemented WE medium showed the least cytotoxicity and most proliferation results after 4 and 6?weeks (as measured by LDH release and MTT assay) (Fig.?S1a, b). The superior qualities of WE medium compared with DMEM under epidermis ALI conditions in terms of preservation of keloid architecture and RNA content were confirmed by histology (Fig.?2). In the DMEM explant group, thinning of the stratum granulosum and papillary dermis became a prominent feature by week 4. This was seen only in some explants cultured in WE by week 6 (Fig.?2a). Pyronin Y histochemistry for measuring RNA preservation was employed, which stains double-stranded nucleic acid, especially RNA (17). Progressive reduction of tissue viability by long-term OC maintenance was marked in the epidermis, which showed a gradual decline in its histochemically detectable RNA content (Fig.?2b) (the detected signals corresponded to RNA was shown by RNAse digestion of sections, which greatly reduced [http://assets.twoorb.com/forum/discussion/336884/azd4547-got-you-way-down-some-of-us-have-the-solution#Item_1 AZD4547 Got You Way Down? Some Of Us Have The Solution] the Pyronin Y signal [negative control]). WE appeared to preserve epidermal RNA content better than DMEM. In the submerged collagen gel matrix explants, instead, the epidermis got progressively dislodged from the underlying dermis and KD tissue at week 6 (data not shown). With the ALI design and in WE medium, even the skin vasculature inside and around the keloid tissue remained reasonably well preserved for at least 4?weeks, as indicated by CD31+ (Table S3) and CD34+ cells (Fig.?S4a). Keloids are well characterized by abundant collagen accumulation, mainly collagen-I and �CIII (25,26). This was well preserved under ALI condition where both tested media could be easily visualized by Herovici��s picropolychrome histochemistry (27). This revealed an abundance of red (collagen-I) and blue (collagen-III) fibres in the reticular dermis (Fig.?2c). In the DMEM group explants, in the first 2?weeks, a distinct layer of blue stained fine fibres existed underneath the epidermis, in which red fibres [http://assets.twoorb.com/forum/discussion/322395/every-thing-you-are-unaware-of-about-isothipendyl#Item_1 Every Thing You Are Unaware Of About isothipendyl] were totally absent. In the DMEM group explants, immature collagen (collagen-III) was replaced with mature collagen (collagen-I) at week 4. However, when the keloid explants were maintained in WE-supplemented media, the explants were able to retain the keloid matrix phenotype for up to 4?weeks (Fig.?2c). Specific immunoreactivity for collagen-I and -III in situ showed 75% of the day 0 (100%) value up to 3?weeks in both DMEM and WE groups, but reduced to 25�C50% of the day 0 level by week 6, in DMEM and WE group, respectively (Table S3).