Journal: Cell Death & Disease

Article Title: MDM2 prevents spontaneous tubular epithelial cell death and acute kidney injury

doi: 10.1038/cddis.2016.390

Figure Lengend Snippet: Tubular injury in tubular cell-specific MDM2 knockout mice kidneys. ( a ) Representative images of PAS-stained kidney sections at a magnification 200x from Pax8rtTA-cre;MDM2f/f and control mice treated for 4, 8 or 11 days with doxycycline showing the progressive aggravation of tubular injury. At day 4 was no tubular damage apparent, at day 8 the tubular cells swelling and vacuolization were the prominent pathologic features and at day 11 the global tubular damage with massive cell loss, tubular dilation and tubular casts was detected. Tubular injury was quantified on PAS-stained renal section as described in Materials and methods. ( b ) Lotus tetragonolobus lectin staining identified proximal tubuli, Tamm-Horsfall protein (THP) staining identified distal tubuli and Aquaporin 2 identified the collecting ducts in Pax8rtTA-cre;MDM2f/f and control mice kidneys treated for 4, 8 or 11 days with doxycycline. The quantitative assessment of tubuli with intact staining patterns is shown for each staining. Data are means±S.E.M. from six mice in each group. * P <0.05, ** P <0.01, *** P <0.005. All images are shown at a magnification of × 100

Article Snippet: For histochemistry we used biotinylated Lotus Tetragonolobus Lectin stain (Vector Labs, Burlingame, CA, USA), Tamm-Horsfall protein stain (Santa Cruz, CA, USA) and rabbit anti-mouse Aquaporin 2 (Abcam, Cambridge, UK).

Techniques: Knock-Out, Staining

Journal: Scientific Reports

Article Title: A fixation method for the optimisation of western blotting

doi: 10.1038/s41598-019-43039-3

Figure Lengend Snippet: Fixation-dependent differences in lectin staining intensities when using PVDF and nitrocellulose membranes. Pooled human serum proteins (3 μg) were separated on 10% SDS-PAGE, and the proteins were transferred onto PVDF and nitrocellulose membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments and followed by staining with lectins (LCA and SNA). Lane i, CBB staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, sample heating at 100 °C; lane v, organic solvent (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) treatments at room temperature; lane vi, organic solvent treatments followed by sample heating at 100 °C. All treatments were applied for 30 min. Left, WB pattern; right, quantitative analysis (n = 3 individual experiments). The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. ** Significantly different p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars).

Article Snippet: Biotinylated lectins, LCA, SNA, PHA-E, PHA-L, and AAL (Supplementary Table ), were purchased from Vector Laboratories Inc. (Burlingame, CA, USA).

Techniques: Staining, SDS Page, Software

Journal: Developmental biology

Article Title: Mi-2/NuRD is required in renal progenitor cells during embryonic kidney development

doi: 10.1016/j.ydbio.2012.11.018

Figure Lengend Snippet: Differentiated epithelial structures are reduced in number in Mi2βΔ/Δ kidneys. (A–N) Immunofluoresence and quantification of differentiated structures in wild type and Mi2βΔ/Δ kidneys at E15.5. NCAM (red) staining of wild type (A) and Mi2βΔ/Δ kidneys (B) revealed a decreased number of renal vesicles (arrowheads) and later differentiated comma- and S-shaped bodies (asterisks) in Mi2βΔ/Δ kidneys (C). A similar reduction (F) was noted in the number of Lhx1-positive (red) differentiated structures in Mi2βΔ/Δ kidneys (E) compared to wild type (D). WT1 (green) staining in wild type (G) and mutants (H) indicates that fewer glomeruli formed in Mi2βΔ/Δ kidneys (I). Glomeruli that formed in mutants are smaller and showed an abnormal arrangement of the podocyte layer (G, H insets). Staining of LTL (green) and E-cadherin (red) in wild type (J) and mutants (K) indicates that proximal and distal nephron segments formed (J, K insets) in Mi2β mutants, but their numbers were reduced (L). E-cadherin-positive distal tubules were distinguished from UB epithelium by co-staining with cytokeratin which stains only UB (M, N).

Article Snippet: Immunofluorescence was performed on 10 µm frozen sections using rabbit anti-Sall1( Kiefer et al., 2002 ), mouse anti-Mi2β (1:500, Abcam), mouse anti-NCAM (1:300, Abcam), rabbit anti-WT1 (1:1000, Santa Cruz) biotin -LTL (1:200, Vector), rat anti-E-cadherin (1:1000, Abcam), rabbit anti-pHH3 (1:1000, Fisher), rabbit anti-Cited1 (1:500, Abcam), rabbit anti-Six2 (1:500, Lifespan Biosciences), rabbit anti-Pax2 (1:2000, Covance), rabbit anti-Lef1 (1:250, Cell Signaling), mouse anti-Lhx1 (1:50, Developmental Studies Hybridoma Bank) and mouse anti-cytokeratin (1:1000, Lifespan Biosciences).

Techniques: Staining