Fig 1: RvD6s enhance corneal nerve regeneration. (A) Whole-mount images of normal corneal nerves stained with anti-PGP9.5, a pan-marker for total corneal nerves and SP, a major neuropeptide in the mouse cornea. The insets, which are marked by a dashed box in the whole-mount images, show the amplified center area of the cornea with double PGP 9.5 and SP staining, and PGP 9.5 and SP alone. (B,C) Representative wholemount images and calculated nerve density of PGP 9.5 (B) and SP (C) positive axons at 12 days after injury and treatment. Data were normalized to the baseline (uninjured corneas in A). The statistical p-value is derived from one-way ANOVA, followed by Tukey’s honest significant difference (HSD) multiple pairwise comparisons.
Fig 2: Western blot images (top) and quantifications (bottom) of UCHL1 in mouse skeletal muscles or C2C12 myotubes. (A): UCHL1 protein levels in slow oxidative muscle soleus and fast glycolytic muscle EDL (n = 4); (B): Immunofluorescent staining for UCHL1 (red) and type I myosine heavy chain (green) in soleus muscle section. The scale bar = 20 µm). (C): UCHL1 protein levels in soleus from fed or fasted mice (n = 3 per group); (D): UCHL1 protein levels in C2C12 myotubes complete media (CM, containing 10% FBS and 4.5 g/L glucose), incomplete media (ICM, containing 4.5 g/L glucose but no FBS), or the media without FBS and glucose (NG). (n = 4 per group).
Fig 3: Western blot images (A) and quantifications (B–E) of muscle samples from WT and UCHL1 smKO mice for key proteins that are involved in lipid metabolisms, including AMPKa ((B), n = 5), Akt ((C), n = 3), SDHA ((D), n = 5), and PHD ((E), n = 3).
Fig 4: Imaging whole mount scWAT(A–H) A series of images captured of whole mount scWAT display various combinations of the immunostaining and imaging described in this protocol. Captured on Nikon E400 (A–C), Leica SP8 (D–F, H), or Nikon A1R (G). Peripheral nerves (green) around autofluorescent lymphatic vessels. Widefield, 10× objective, 250 µm scale bar, BL6 mouse, anti-PGP9.5 (A). Sympathetic nerves (magenta). Widefield, 40× objective, 50 µm scale bar, BL6 mouse, anti-Tyrosine Hydroxylase (B). Peripheral nerve bundle (green) and blood vessels (red). Widefield, 40× objective, 250 µm scale bar, BL6 mouse, anti-PGP9.5 and Isolectin-IB4 (C). Peripheral nerve bundle (cyan) co-stained for sympathetic nerves (magenta). Confocal Zmax projection, 20× objective, 145.5 µm scale bar, PGP9.5-EGFP mouse, anti-Tyrosine Hydroxylase (D). Sympathetic nerves (orange) around a blood vessel (not stained). Confocal Zmax projection, 63× objective, 46.2 µm scale bar BL6 mouse, anti-Tyrosine Hydroxylase (E). Peripheral nerves (height-color coded) around a blood vessel (not stained). Confocal Zmax projection, 63× objective, 50 µm scale bar, BL6 mouse, anti-ß3-Tubulin (F). Peripheral nerves (green), capillaries (red), nuclei (blue). Confocal Zmax projection, 20× objective, 100 µm scale bar PGP9.5-EGFP mouse, DAPI and Isolectin (G). Intact inguinal scWAT depot innervation (grey). Confocal Zmax projection, 5× objective, 16.6 mm scale bar, tiled whole depot, BL6 mouse, anti-PGP9.5 (H).
Fig 5: Z-depth reduction (ZDR) and tissue morphology(A) Axillary subcutaneous white adipose tissue (axi-scWAT) and inguinal (ing-scWAT) depots were Z-depth reduced and mounted on slides.(B) Tissue area was measured before ZDR (control), after ZDR, and after mounting. N = 3, paired two-tailed Student’s t-test, alpha level 0.05, error bars are SEMs.(C) Tissue thickness in the z-axis was measured at the thickest point before and after ZDR and mounting. Mounted tissues were measured by 3D projecting tissue autofluorescence captured on a Nikon A1R with a 20X objective. N = 3, paired two-tailed Student’s t-test, alpha level 0.05, error bars are SEMs.(D) Ing-scWAT was either whole mount processed (Z-depth reduced) or left unaltered (control) and placed on a slide and imaged with transmitted light. Four representative 10X micrographs were captured for each tissue and the area for 30–120 cells was measured per micrograph and analyzed using a two-tailed Student’s t-test, Alpha level 0.05, error bars are SEMs.(F) Hematoxylin staining of 7 µm ing-scWAT sections that either received ZDR before paraffin embedding or did not (control). Both ing-scWAT depots were excised from a PGP9.5+/- (green) direct reporter mouse (C57BL/6-Tg(Uchl1-EGFP)G1Phoz/J) and co-stained with IB4 (vasculature, red) and DAPI (nuclei, blue). One depot was Z-depth reduced and mounted whereas the other was left uncompressed to demonstrate morphological changes introduced by compressing the tissue; demonstrated through 3D reconstructions of similar structures.Other details: Transmitted light micrographs were captured on a Nikon E400 upright microscope (D and E). Confocal micrographs captured on Nikon A1R (C and F). Scale bars are 186 µm and 174 µm (C), 200 µm (D and F) and 50 µm (E).
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