Human Serum Albumin Antibody

Detection of Albumin in HepG2 Human Cell Line by Flow Cytometry.

HepG2 human hepatocellular carcinoma cell line was stained with Mouse Anti-Human Serum Albumin Monoclonal Antibody (Catalog # MAB1455, filled histogram) or isotype control antibody (Catalog # MAB003, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.

Detection of Human Albumin by Simple Western^TM.

Simple Western lane view shows lysates of human serum, loaded at 1:25000. A specific band was detected for Albumin at approximately 64 kDa (as indicated) using 1 µg/mL of Mouse Anti-Human Serum Albumin Monoclonal Antibody (Catalog # MAB1455) . This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human Albumin by Simple Western^TM.

Simple Western lane view shows lysates of human liver tissue, loaded at 0.2 mg/mL. A specific band was detected for Albumin at approximately 64 kDa (as indicated) using 10 µg/mL of Mouse Anti-Human Serum Albumin Monoclonal Antibody (Catalog # MAB1455). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Serum Albumin in Human Liver Using Dual RNAscope^®ISH and IHC.

Serum Albumin mRNA was detected in formalin-fixed paraffin-embedded tissue sections of human liver probed with ACD RNAScope^®Probe (Catalog # 600941) and stained using ACD RNAscope^®2.5 HD Detection Reagents-Red (top image, Catalog # 32260). Adjacent tissue section was processed for immunohistochemistry using R&D Systems Mouse Anti-Human Serum Albumin Monoclonal Antibody (Catalog # MAB1455) at 0.05 ug/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte HRP Polymer Antibody (R&D Systems, Catalog # VC001) and DAB chromogen (lower image, yellow-brown). Tissues were counterstained with hematoxylin (blue).

Detection of Mouse Albumin by Immunohistochemistry

Heterochronic blood exchange effects on muscle regeneration and performance.One day after blood exchange mice were injured by intramuscular injections of CTX into TA. Five days after injury, TA muscles were isolated, cryo-sectioned and analysed. (a) TA muscles from young mice receiving young blood (YY), young mice receiving old blood (YO), old mice receiving young blood (OY) and old mice receiving old blood (OO) were analysed by haematoxylin and eosin (H&E) staining and immunofluorescence with anti-eMyHC antibody. Representative images show an injury site and nascent de-novo formed eMyHC+ myofibers which are smaller in size with central nuclei than uninjured myofibers. Scale bar, 50 μm for H&E panel and 25 μm for immunofluorescence panel. (b,c) Regeneration indices ±s.e.m. were quantified from H&E images (b) and eMyHC images (c) by counting the number of nascent de-novo formed myofibers and dividing by the total number of nuclei present at the injury/regeneration site. By H&E: *P<0.05 N=4 per group. Significant students t test differences exist between YO and OY (P=0.045), YY and OY (P=0.043), YY and OO (P=0.0004), YO and OO (P=0.0042) and between OY and OO (P=0.015). By eMyHC: *P<0.05, N=4 per group; OY and OO P=0.041, YY and OO P=0.00009, and YO to OO P=0.001. (d) Fibrotic/inflammatory indexes were quantified as total injury area minus regenerated myofiber area, per injury site, using the H&E images15. T-test **P<0.005, n=3–4 per group. Muscle from old to old isochronic exchange had diminished regenerative capacity and more fibrosis, as compared with muscle from young to young isochronic exchange. Heterochronic blood exchange significantly improved regeneration of old muscle after experimental injury and reduced fibrosis, but no significant decline in young muscle regeneration was seen. (e) A four-limb hanging test was conducted with isochronically and heterochronically transfused mice that were not injured, before and at 6 days after the blood exchange. Maximal hanging time was multiplied by body weight (hang index). T-test n=4–8, P=0.01 YY post transfusion compared with O training, and YO, OY, OO post-transfusion performance. Y to O training and YO, OY and OO were NS=not statistically different. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27874859), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Albumin by Immunohistochemistry

Heterochronic blood exchange effects on hepatogenesis and liver fibrosis and adiposity.(a) Livers from YY, YO, OY and OO mice with and without experimental muscle injury as above were cryo-sectioned at 10 μm and immuno-stained for Ki67 (red), hepatocyte marker albumin (green) and Hoechst (blue). Representative images show YY livers with and without injury. Scale bar, 50 μm. (B&C. Quantification of hepatocyte proliferation was by counting the average number of Ki67+,abumin+,Hoechst+ cells per 10 μm section from multiple sections of each blood exchange cohort. (b) Old hepatocyte showed increased proliferation and young hepatocytes showed less proliferation with heterochronic blood as compared with isochronic blood exchanges in animals with injured muscle (t test P=0.00028). (c) This trend continues without muscle injury, but the total numbers of proliferating hepatocytes decline by twofold, (P=0.02411). *P<0.05; **P<0.005; n=3–5. (d) As previously published4, there were fibrotic clusters exclusively in the old livers of small Ki67+ve, albumin negative Ki67+ cells. Scale bar, 50 μm, × 40 magnification. (e,f) Fibrotic index was calculated as the average number of albumin negative proliferative cell clusters per four 10 μm sections. The fibrotic index diminished in old mice exchanged with young blood with muscle injury (e) (t test P=0.048 N=4, *P<0.05) or without (f) (t test P=0.00776. N=3; **P<0.005). (g) Liver adiposity was assayed by Oil Red in 10 μm cryosections. Shown are representative images acquired at × 20 magnification. (h). Liver adiposity (red) was quantified by Image J, dramatically increased with age and was attenuated by young blood in old mice (t test N=3, P=0.022), while adiposity remained unchanged in young mice that were transfused with the old blood (see Supplementary Figure 4). Shown are means±s.e.m. for all histograms. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27874859), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Albumin by Immunohistochemistry

Heterochronic blood exchange effects on hepatogenesis and liver fibrosis and adiposity.(a) Livers from YY, YO, OY and OO mice with and without experimental muscle injury as above were cryo-sectioned at 10 μm and immuno-stained for Ki67 (red), hepatocyte marker albumin (green) and Hoechst (blue). Representative images show YY livers with and without injury. Scale bar, 50 μm. (B&C. Quantification of hepatocyte proliferation was by counting the average number of Ki67+,abumin+,Hoechst+ cells per 10 μm section from multiple sections of each blood exchange cohort. (b) Old hepatocyte showed increased proliferation and young hepatocytes showed less proliferation with heterochronic blood as compared with isochronic blood exchanges in animals with injured muscle (t test P=0.00028). (c) This trend continues without muscle injury, but the total numbers of proliferating hepatocytes decline by twofold, (P=0.02411). *P<0.05; **P<0.005; n=3–5. (d) As previously published4, there were fibrotic clusters exclusively in the old livers of small Ki67+ve, albumin negative Ki67+ cells. Scale bar, 50 μm, × 40 magnification. (e,f) Fibrotic index was calculated as the average number of albumin negative proliferative cell clusters per four 10 μm sections. The fibrotic index diminished in old mice exchanged with young blood with muscle injury (e) (t test P=0.048 N=4, *P<0.05) or without (f) (t test P=0.00776. N=3; **P<0.005). (g) Liver adiposity was assayed by Oil Red in 10 μm cryosections. Shown are representative images acquired at × 20 magnification. (h). Liver adiposity (red) was quantified by Image J, dramatically increased with age and was attenuated by young blood in old mice (t test N=3, P=0.022), while adiposity remained unchanged in young mice that were transfused with the old blood (see Supplementary Figure 4). Shown are means±s.e.m. for all histograms. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27874859), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Albumin by Immunocytochemistry/Immunofluorescence

Differentiation of hESCs into HLCs.a The relative hepatocyte (ALB, AAT, CK18, and ASGPR1), cholangiocyte (SOX9), and hepatoblast (AFP) gene expression levels of day 19 differentiated cells with different treatments (groups A and B) were determined by qPCR. HGF (hepatocyte growth factor, 20 ng/mL); OSM (oncostatin M, 20 ng/mL); Dex (dexamethasone, 10 µM); SB431542 (TGF beta inhibitor, 2 µM); and RO4929097 (Notch inhibitor, 1 µM). b Immunofluorescence analysis of ALB, AAT, ASGPR1, and CK18 expression in group B-induced differentiated cells on day 19. c The expression levels of ALB and ASGPR1 in group B-induced differentiated cells were determined by flow cytometry on day 19. Isotype control antibodies were used as controls. d The relative hepatocyte (ALB, AAT, CK18, ASGPR1, and AFP) gene expression levels of differentiated HLCs (group B) compared with those of hESCs and primary human hepatocytes (PHHs) were determined by qPCR. e Albumin secretion of HLCs (black line) on days 12, 14, 16, 18, and 20 and PHHs (dotted line) on day 2 were determined by ELISA. *p < 0.05, **p < 0.01; data are represented as the mean ± SD. Scale bar, 50 µm Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31601782), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Albumin by Immunohistochemistry

NLRP3 inflammasome-dependent pyroptosis occurs in liver fibrosis. (A) IHC staining for GSDMD, IL-1 beta, and IL-18 in liver sections from liver fibrosis patients and HCs. Scale bar: 40 µm. (B–D) ELISA analyses of serum levels of GSDMD (B), IL-1 beta (C), and IL-18 (D) in liver fibrosis patients (n = 89) and HCs (n = 60). (E) Representative immunofluorescence images of NLRP3 (red) and albumin (hepatocyte marker) (top), F4/80 (KC marker) (middle) or alpha-SMA (HSC marker) (bottom) (green) from the human fibrotic liver tissues. Scale bar: 40 µm. (F) Schematic diagram of the study. Liver fibrosis was induced by CCl4 injection for 8 weeks. (G) Representative mouse liver histology of H&E, Sirius Red staining, and IHC staining for alpha-SMA, GSDMD, and IL-1 beta. Black scale bar: 100 µm; Red scale bar: 50 µm. (H–J) ELISA analyses for serum levels of GSDMD (H), IL-1 beta (I), and IL-18 (J) in CCl4 group mouse (n = 5) and vehicle group mouse (n = 5). (K) Representative immunofluorescence images of NLRP3 (red) and albumin (hepatocyte marker) (top), F4/80 (KC marker) (middle) or alpha-SMA (HSC marker) (bottom) (green) from the 8-week CCl4-treated mouse liver. The vehicle group mouse liver was used as a control. Scale bar: 40 µm. (L) The qRT-PCR analysis for mRNA levels of IL-1 beta in THP-1 macrophages treated with LPS to induce pyroptosis. (M) ELISA analysis for IL-1 beta expression in supernatants from THP-1. (N) Western blot analysis of COL1A1, alpha-SMA, and TGF-beta expression in LX-2 cells which were exposed to CM from LPS-treated THP-1 macrophages. The protein expression was quantified by densitometry and normalized to beta-actin and are shown as fold changes relative to the control group (right panel). ** p < 0.01, *** p < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36429008), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Albumin by Immunohistochemistry

NLRP3 inflammasome-dependent pyroptosis occurs in liver fibrosis. (A) IHC staining for GSDMD, IL-1 beta, and IL-18 in liver sections from liver fibrosis patients and HCs. Scale bar: 40 µm. (B–D) ELISA analyses of serum levels of GSDMD (B), IL-1 beta (C), and IL-18 (D) in liver fibrosis patients (n = 89) and HCs (n = 60). (E) Representative immunofluorescence images of NLRP3 (red) and albumin (hepatocyte marker) (top), F4/80 (KC marker) (middle) or alpha-SMA (HSC marker) (bottom) (green) from the human fibrotic liver tissues. Scale bar: 40 µm. (F) Schematic diagram of the study. Liver fibrosis was induced by CCl4 injection for 8 weeks. (G) Representative mouse liver histology of H&E, Sirius Red staining, and IHC staining for alpha-SMA, GSDMD, and IL-1 beta. Black scale bar: 100 µm; Red scale bar: 50 µm. (H–J) ELISA analyses for serum levels of GSDMD (H), IL-1 beta (I), and IL-18 (J) in CCl4 group mouse (n = 5) and vehicle group mouse (n = 5). (K) Representative immunofluorescence images of NLRP3 (red) and albumin (hepatocyte marker) (top), F4/80 (KC marker) (middle) or alpha-SMA (HSC marker) (bottom) (green) from the 8-week CCl4-treated mouse liver. The vehicle group mouse liver was used as a control. Scale bar: 40 µm. (L) The qRT-PCR analysis for mRNA levels of IL-1 beta in THP-1 macrophages treated with LPS to induce pyroptosis. (M) ELISA analysis for IL-1 beta expression in supernatants from THP-1. (N) Western blot analysis of COL1A1, alpha-SMA, and TGF-beta expression in LX-2 cells which were exposed to CM from LPS-treated THP-1 macrophages. The protein expression was quantified by densitometry and normalized to beta-actin and are shown as fold changes relative to the control group (right panel). ** p < 0.01, *** p < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36429008), licensed under a CC-BY license. Not internally tested by R&D Systems.