rabbit anti human tim 4  (Proteintech)

Journal: bioRxiv

Article Title: Loss of TIM4-Dependent Efferocytosis in Kupffer Cells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis

doi: 10.1101/2024.01.30.578023

Figure Lengend Snippet: ( A ) Immunostaining of human liver sections from subjects with a NAS score of 0-2 or 3-6 using TUNEL (cell death) and anti-CD68 (macrophage, Mϕ). Arrows indicate macrophage-associated TUNEL + cells, while arrowheads indicate macrophage-free TUNEL+ cells. Efferocytosis was determined by calculating the ratio of macrophage-associated: free TUNEL + cells and total TUNEL + cells per field (n = 4 in the NAS 0-2 group and 13 in the NAS 3-6 group). ( B ) Livers from mice fed the HF-CDAA diet for the indicated weeks were assayed for efferocytosis, using anti-cleaved caspase-3 (cl-Casp3) to mark apoptotic cells and anti-Mac2 to stain macrophages (n=3-9 mice per group. Examples of images at 3 and 12 weeks of diet feeding are shown. Bar, 100 μm. Arrows indicate macrophage-associated apoptotic cells, while arrowheads indicate macrophage free apoptotic cells. ( C ) Immunoblots of TIM4 in another cohort of human normal and NASH liver, with data quantification (n = 7/group). ( D ) Immunostaining of human normal and NASH liver sections from panel A using anti-TIM4 (red) and anti-CD68 (green). Bar, 50 μm. Data were quantified as TIM4 MFI in CD68 + macrophages relative to normal liver (n = 5 in normal group and n = 7 in NASH group). For immunofluorescence images, nuclei are stained with DAPI (blue). All data are means ± SEM. *p <0.05, **p <0.01, ***p <0.001 by Student’s t-test (panels A, C, and D) or one-way ANOVA (panel B).

Article Snippet: For immunofluorescence experiments, human liver sections were incubated at 4 °C overnight with anti-human TIM4 (Cell signaling technology, #75484T, RRID:AB_2799871, 1:200 dilution), anti-CD68 (Agilent, #GA60961-2, RRID:AB_2661840, 1:500 dilution), and anti-cleaved caspase3 (Cell signaling technology, # 9661, RRID:AB_2341188, 1:100 dilution) in PBS containing 1% donkey serum.

Techniques: Immunostaining, TUNEL Assay, Staining, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of TIM4-Dependent Efferocytosis in Kupffer Cells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis

doi: 10.1101/2024.01.30.578023

Figure Lengend Snippet: ( A ) Images of primary human KCs stained for F-actin (red) after exposure to apoptotic human hepatocytes (apHCs, green) for 6 h in the presence of IgG or anti-TIM4. Bar, 10 μm. The data were quantified as both the average volume of engulfed cargo and the MFI of internalized apHCs (n = 7 biological replicate cells/group). ( B ) Quantification of efferocytosis by primary mouse KCs after exposure to PKH67-labaled apoptotic mouse hepatocytes (n = 4 biological replicate cells/group). ( C-I ) Mice were fed the HF-CDAA NASH diet for 4 weeks and treated with IgG or anti-TIM4 during weeks 3-4 (n = 5 mice/group). ( C ) Images of liver sections immunostained with anti-Mac2 (green) and anti-cl-Casp3 (red). Bar, 25 μm. Arrows depict efferocytic macrophage, while arrowheads depict macrophage-free apoptotic cells. Efferocytosis was quantified as in . ( D ) Staining and quantification of picrosirius red–positive area. Bar, 200 μm. ( E ) Immunostaining and quantification of collagen 1a1 (COL1A1)-positive area. Bar, 200 μm. ( F ) Immunostaining and quantification of α-SMA-positive area. Bar, 50 μm. ( G ) Immunostaining and quantification of osteopontin (OPN)-positive area. Bar, 200 μm. ( H ) Immunostaining and quantification of cytokeratin 19 (CK19)-positive area. Bar, 200 μm. ( I ) Immunostaining of F4/80, with quantification of F4/80 + hepatic crown-like structures (hCLS)/field. Bar, 200 μm. For immunofluorescence images, nuclei are stained with DAPI (blue). All data are means ± SEM. *p <0.05, **p <0.01 by Student’s t-test.

Article Snippet: For immunofluorescence experiments, human liver sections were incubated at 4 °C overnight with anti-human TIM4 (Cell signaling technology, #75484T, RRID:AB_2799871, 1:200 dilution), anti-CD68 (Agilent, #GA60961-2, RRID:AB_2661840, 1:500 dilution), and anti-cleaved caspase3 (Cell signaling technology, # 9661, RRID:AB_2341188, 1:100 dilution) in PBS containing 1% donkey serum.

Techniques: Staining, Immunostaining, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of TIM4-Dependent Efferocytosis in Kupffer Cells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis

doi: 10.1101/2024.01.30.578023

Figure Lengend Snippet: For panels A-H , Timd4 fl/fl (control, Ctr) and Timd4 fl/fl ; Clec4f-Cre +/- (knockdown, KD) male mice were fed the HF-CDAA diet for 4 weeks (n = 8-10 mice/group). ( A ) Immunostaining of liver sections using anti-TIM4 (red) and anti-F4/80 (green). Data were quantified as macrophage TIM4 MFI relative to control mice (n = 4 mice/group). Bar, 50 μm. ( B ) Images of liver sections immunostained with anti-Mac2 (green) and anti-cl-Casp3 (red). Bar, 50 μm. Arrows depict efferocytic macrophages, while arrowheads depict macrophage-free apoptotic cells. Efferocytosis was quantified as in . ( C ) Staining and quantification of picrosirius red–positive area (arrows). Bar, 200 μm. ( D ) Immunostaining and quantification of collagen 1a1-positive area. Bar, 200 μm. ( E ) Immunostaining and quantification of α-SMA-positive area. Bar, 50 μm. ( F ) Immunostaining and quantification of OPN-positive area. Bar, 200 μm. ( G ) Immunostaining and quantification of CK19-positive area. Bar, 200 μm. ( H ) Immunostaining of F4/80, with quantification of F4/80 + hepatic crown-like structures/field. Bar, 200 μm. For panels I-K , Timd4 fl/fl (control, Ctr) and Timd4 fl/fl ; Clec4f-Cre +/- (knockdown, KD) mice were fed the HF-CDAA diet for 8 weeks (n = 13 mice/group). ( I ) Images of liver sections immunostained with anti-Mac2 (green) and anti-cl-Casp3 (red). Bar, 100 μm. The arrows depicts an efferocytic macrophage, and the arrowheads depict macrophage-free apoptotic cells. Efferocytosis was quantified as in . ( J ) Staining and quantification of picrosirius red–positive area (arrows). Bar, 200 μm. ( K ) Immunostaining and quantification of collagen 1a1-positive area. Bar, 200 μm. ( L ) Immunostaining and quantification of osteopontin (OPN)-positive area. Bar, 200 μm. For immunofluorescence images, nuclei are stained with DAPI (blue). All data are means ± SEM. *p <0.05, **p <0.01 by Student’s t-test.

Article Snippet: For immunofluorescence experiments, human liver sections were incubated at 4 °C overnight with anti-human TIM4 (Cell signaling technology, #75484T, RRID:AB_2799871, 1:200 dilution), anti-CD68 (Agilent, #GA60961-2, RRID:AB_2661840, 1:500 dilution), and anti-cleaved caspase3 (Cell signaling technology, # 9661, RRID:AB_2341188, 1:100 dilution) in PBS containing 1% donkey serum.

Techniques: Control, Knockdown, Immunostaining, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of TIM4-Dependent Efferocytosis in Kupffer Cells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis

doi: 10.1101/2024.01.30.578023

Figure Lengend Snippet: Timd4 fl/fl (control, Ctr) and Timd4 fl/fl ; Clec4f-Cre (knockdown, KD) male mice were fed the FPC diet for 16 weeks (n = 8-9 mice/group). ( A ) Immunostaining of liver sections using anti-TIM4 (red) and anti-F4/80 (green). Data were quantified as macrophage TIM4 MFI relative to control mice (n = 5 mice/group). Bar, 50 μm. ( B ) Images of liver sections immunostained with anti-Mac2 (green) and anti-cl-Casp3 (red). Bar, 50 μm. The arrow depicts an efferocytic macrophage, and the arrowheads arrows depict macrophage-free apoptotic cells. Efferocytosis was quantified as in . ( C ) Staining and quantification of picrosirius red–positive area (arrows). Bar, 50 μm. ( D ) Immunostaining and quantification of COL1A1-positive area. Bar, 200 μm. ( E ) Immunostaining and quantification of α-SMA-positive area. Bar, 50 μm. ( F ) Immunostaining and quantification of OPN-positive area. Bar, 200 μm. ( G ) Liver mRNAs for the indicated fibrogenic genes, expressed as relative to the values for control mice. ( H ) Immunostaining and quantification of CK19-positive area. Bar, 200 μm. ( I ) Immunostaining of F4/80, with quantification of hepatic crown-like structures/field. Bar, 200 μm. All data are means ± SEM. *p <0.05, **p <0.01, ***p <0.001 by Student’s t-test.

Article Snippet: For immunofluorescence experiments, human liver sections were incubated at 4 °C overnight with anti-human TIM4 (Cell signaling technology, #75484T, RRID:AB_2799871, 1:200 dilution), anti-CD68 (Agilent, #GA60961-2, RRID:AB_2661840, 1:500 dilution), and anti-cleaved caspase3 (Cell signaling technology, # 9661, RRID:AB_2341188, 1:100 dilution) in PBS containing 1% donkey serum.

Techniques: Control, Knockdown, Immunostaining, Staining

Journal: bioRxiv

Article Title: Loss of TIM4-Dependent Efferocytosis in Kupffer Cells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis

doi: 10.1101/2024.01.30.578023

Figure Lengend Snippet: Control mice (TRE-TIMD4 and CD68rtTA; Ctr; n = 15) and inducible CD68rtTA:TRE-TIMD4 transgenic mice (iTg, n = 13) were fed the HF-CDAA diet for 10 weeks, with 75 μg/ml doxycycline (Dox) added to the drinking water during weeks 6-10. ( A ) Immunostaining of liver sections using anti-TIM4 (red) and anti-Mac2 (green). Data were quantified as macrophage TIM4 MFI relative to control mice. Bar, 50 μm. ( B ) Images of liver sections immunostained with anti-Mac2 (green) and anti-cl-Casp3 (red). Bar, 25 μm. Arrows depict macrophage-free apoptotic cells. Efferocytosis was quantified as in . ( C ) Staining and quantification of picrosirius red– positive area (arrows). Bar, 200 μm. ( D ) Immunostaining and quantification of COL1A1-positive area. Bar, 200 μm. ( E ) Immunostaining and quantification of α-SMA-positive area. Bar, 100 μm. ( F ) Immunostaining and quantification of OPN-positive area. Bar, 100 μm. ( G ) Immunostaining and quantification of CK19-positive area. Bar, 200 μm. ( H ) Immunostaining of F4/80, with quantification of hepatic crown-like structures/field. Bar, 200 μm. All data are means ± SEM. *p <0.05, **p <0.01, ***p <0.001 by Student’s t-test.

Article Snippet: For immunofluorescence experiments, human liver sections were incubated at 4 °C overnight with anti-human TIM4 (Cell signaling technology, #75484T, RRID:AB_2799871, 1:200 dilution), anti-CD68 (Agilent, #GA60961-2, RRID:AB_2661840, 1:500 dilution), and anti-cleaved caspase3 (Cell signaling technology, # 9661, RRID:AB_2341188, 1:100 dilution) in PBS containing 1% donkey serum.

Techniques: Control, Transgenic Assay, Immunostaining, Staining

Journal: bioRxiv

Article Title: Loss of TIM4-Dependent Efferocytosis in Kupffer Cells Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis

doi: 10.1101/2024.01.30.578023

Figure Lengend Snippet: ( A-H ) Control mice (TRE-TIMD4 and CD68rtTA; Ctr; n = 17) and inducible CD68rtTA:TRE-TIMD4 transgenic mice (iTg, n = 8) were fed the FPC diet for 16 weeks, with 75 μg/ml doxycycline (Dox) added to the drinking water during weeks 9-16. ( A ) Immunostaining of liver sections using anti-TIM4 (red) and anti-Mac2 (green). Data were quantified as macrophage TIM4 MFI relative to control mice (n = 3 mice/group). Bar, 50 μm. ( B ) Images of liver sections immunostained with anti-Mac2 (green) and anti-cl-Casp3 (red). Bar, 100 μm. Arrows depict efferocytic macrophages, and the arrowhead depicts a macrophage-free apoptotic cell. Efferocytosis was quantified as in . ( C ) Staining and quantification of picrosirius red–positive area (arrows). Bar, 100 μm. ( D ) Immunostaining and quantification of COL1A1-positive area. Bar, 200 μm. ( E ) Immunostaining and quantification of α-SMA-positive area. Bar, 50 μm. ( F ) Immunostaining and quantification of OPN-positive area. Bar, 200 μm. ( G ) Liver mRNAs for the indicated fibrogenic genes, expressed as relative to the values for control mice. ( H ) Immunostaining and quantification of CK19-positive area. Bar, 200 μm. ( I ) Immunostaining of F4/80, with quantification of F4/80 + crown-like structures/field. Bar, 200 μm. All data are means ± SEM. *p <0.05, **p <0.01, ***p <0.001 by Student’s t-test.

Article Snippet: For immunofluorescence experiments, human liver sections were incubated at 4 °C overnight with anti-human TIM4 (Cell signaling technology, #75484T, RRID:AB_2799871, 1:200 dilution), anti-CD68 (Agilent, #GA60961-2, RRID:AB_2661840, 1:500 dilution), and anti-cleaved caspase3 (Cell signaling technology, # 9661, RRID:AB_2341188, 1:100 dilution) in PBS containing 1% donkey serum.

Techniques: Control, Transgenic Assay, Immunostaining, Staining