fluorescein conjugated vicia villosa lectin  (Vector Laboratories)

Journal: Disease Models & Mechanisms

Article Title: Long-chain polyphosphates induce glomerular microthrombi and exacerbate LPS-induced acute kidney injury in mouse

doi: 10.1242/dmm.052361

Figure Lengend Snippet: Kidney tubular apoptosis and expression of the inflammatory markers cJUN and IRF1 in PolyP and/or LPS-treated mice. Mice were treated with medium-chain (P100) or long-chain (P700) PolyPs alone, or in combination with LPS (LPS+P100 or LPS+P700, respectively). (A) Immunofluorescence staining of apoptotic cells against cleaved caspase 3 (Casp3, red) and of kidney proximal tubule brush borders with lotus tetragonolobus lectin (LTL, green). Apoptosis inside disrupted proximal tubules was seen in mice treated with P700, most prominently in the outer medulla. In LPS+P700-treated mice several individual apoptotic tubular and glomerular cells were additionally seen in affected foci within the cortex. Only few apoptotic cells were seen in LPS-and LPS+P100-treated groups. (B) The kidney cortex showed distinct cJUN-positive (red) foci in P700-treated mice, which were located adjacent to the glomeruli with a granular nephrin (green) phenotype. Mice treated with LPS, LPS+P100 or LPS+P700 showed evenly distributed cJUN protein expression throughout the cortex, independent of the glomeruli with a granular nephrin phenotype. (C) Foci with IRF1-positive (red) nuclei were slightly increased in the kidney cortex in response to treatment with P700 or LPS compared to controls. Strong IRF1-expression was seen in pyknotic nuclei in necrotic areas of LPS+P700-treated mice. Images were acquired using a 20× objective. Boxed areas below each main image show individual staining (left, middle) and merged images (right). Nuclei were stained with Hoechst 33258 (blue). All scale bars: 100 µm.

Article Snippet: Secondary antibodies (anti-rabbit IgG Alexa Fluor 594; Thermo Fisher Scientific, cat. no.: A-21207 and anti-Guinea Pig IgG Alexa Fluor 488, cat. no.: A-11073; both diluted 1:500) or fluorescein-conjugated lotus tetragonolobus lectin (LTL; Vector Laboratories; see ) were incubated for 1 h at room temperature before the slides were washed three times for 15 min. Staining of nuclei with Hoechst 33258 (Thermo Fisher; cat. no.: H1398) was included during the secondary antibody incubation.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Disease Models & Mechanisms

Article Title: Long-chain polyphosphates induce glomerular microthrombi and exacerbate LPS-induced acute kidney injury in mouse

doi: 10.1242/dmm.052361

Figure Lengend Snippet: Glomerular microthrombi consisting of fibrin/collagen, von Willebrand factor and platelet accumulation in the kidney cortex of PolyP and/or LPS-treated mice. Mice were treated with medium-chain (P100) or long-chain (P700) PolyPs alone, or in combination with LPS (LPS+P100 or LPS+P700, respectively). (A) Masson's trichrome stain shows red-stained deposits indicating fibrin (white arrowheads) and blue-stained deposits indicating collagen (blue arrowheads) in the glomerular capillary loops of P700- and LPS+P700-treated mice. Images extracted from a 40× whole-slide scan. Scale bar: 20 µm. (B) Immunofluorescence staining of platelets with antibody against CD61 (red) and of kidney proximal tubule brush borders with lotus tetragonolobus lectin (LTL, green) show platelet accumulation in glomeruli and arterioles of P700- and LPS+P700-treated mice. Images were acquired using a 20× objective. Boxed areas below each main image show individual staining (left, middle) and merged images (right). Nuclei were stained with Hoechst 33258 (blue). Scale bar: 100 µm. (C) Immunofluorescence staining showing von Willebrand factor-positive (vWF, red) deposits in the glomerular capillary loops of P700 and LPS+P700-treated mice, with the podocyte marker, nephrin (green) showing a granular phenotype. Images were acquired using a 100× objective. Magnified views are shown in the top left of each image. Nuclei were stained with Hoechst 33258 (blue). Scale bar: 20 µm.

Article Snippet: Secondary antibodies (anti-rabbit IgG Alexa Fluor 594; Thermo Fisher Scientific, cat. no.: A-21207 and anti-Guinea Pig IgG Alexa Fluor 488, cat. no.: A-11073; both diluted 1:500) or fluorescein-conjugated lotus tetragonolobus lectin (LTL; Vector Laboratories; see ) were incubated for 1 h at room temperature before the slides were washed three times for 15 min. Staining of nuclei with Hoechst 33258 (Thermo Fisher; cat. no.: H1398) was included during the secondary antibody incubation.

Techniques: Staining, Immunofluorescence, Marker

Journal: Disease Models & Mechanisms

Article Title: Long-chain polyphosphates induce glomerular microthrombi and exacerbate LPS-induced acute kidney injury in mouse

doi: 10.1242/dmm.052361

Figure Lengend Snippet: Expression of kallikrein–kinin-system components and tissue factor in kidney of PolyP and/or LPS-treated mice. Mice were treated with medium-chain (P100) or long-chain (P700) PolyPs alone, or in combination with LPS (LPS+P100 or LPS+P700, respectively) (A) Top: Immunofluorescence staining for the tissue kallikrein inhibitor kallistatin (red) and the proximal tubule marker lotus tetragonolobus lectin (LTL, green) shows distinct foci in the outer medulla of the kidney, where both kallistatin protein expression and the proximal tubule brush border integrity are lost in the P700 and LPS+P700-treated mice. Bottom: The renal cortex region of LPS+P700-treated mice displayed augmented kallistatin protein expression in dilated luminal compartments in the necrotic areas, where proximal tubule brush border integrity is lost. Images were acquired using a 20× objective. Boxed areas below each main image show individual staining (left, middle) and merged images (right). Nuclei were stained with Hoechst 33258 (blue). Scale bars: 100 µm. (B) Increased expression of BDKRB2 (red) was seen at tubular cell nuclear membranes in all treatment groups compared to controls. Cytoplasmic aggregation of BDKRB2 was seen in the necrotic areas of P700 and LPS+P700-treated groups. Proximal tubule brush borders were stained with LTL (green), nuclei with Hoechst 33258 (blue). Images were acquired using a 20× objective (top) or a 100× objective (bottom). Scale bars: 100 µm. (C) Transcription levels of Kng2 (encoding kininogen 2 in mouse) and F3 (encoding tissue factor) in kidney tissue measured by RT-qPCR. Red dots indicate the relative gene expression of each mouse compared to the control group. Black dots indicate the geometric mean per group, scaled as one in the control group. Benjamini-Hochberg-adjusted significance levels were calculated using the pairwise Mann–Whitney U -test (significance levels: one symbol P <0.05, two symbols P <0.01, three symbols P <0.001). Symbols used for significance: #, significance compared to the control group; $, significance compared to the LPS-treated group. ns: non-significant.

Article Snippet: Secondary antibodies (anti-rabbit IgG Alexa Fluor 594; Thermo Fisher Scientific, cat. no.: A-21207 and anti-Guinea Pig IgG Alexa Fluor 488, cat. no.: A-11073; both diluted 1:500) or fluorescein-conjugated lotus tetragonolobus lectin (LTL; Vector Laboratories; see ) were incubated for 1 h at room temperature before the slides were washed three times for 15 min. Staining of nuclei with Hoechst 33258 (Thermo Fisher; cat. no.: H1398) was included during the secondary antibody incubation.

Techniques: Expressing, Immunofluorescence, Staining, Marker, Quantitative RT-PCR, Gene Expression, Control, MANN-WHITNEY