Immunohistochemical Analysis of Liver Inflammation

Liver tissues were embedded in paraffin, and slides were cut from the paraffin tissue. Slides were heated with sodium citrate for antigen retrieval and then incubated with primary antibodies including TLR2 (#GB11554, 1:300, Servicebio, Wuhan, China), NLRP3 (#YT5382, 1:100, ImmunoWay Biotechnology), ASC (#YT0365, 1:200, ImmunoWay Biotechnology), Caspase-1 (#GB11383, 1:500, Servicebio), and IL-1β (#GB11113, 1:250, Servicebio) overnight at 4°C. Secondary antibody HRP-conjugated Goat Anti-Rabbit IgG(H+L) (#GB23303, 1:200, Servicebio) was incubated for 50 min at room temperature. After PBS washing, peroxidase substrate DAB (#K5007, DaKo, Copenhagen, Denmark) was used as chromogen. The slices were stained, differentiated, and sealed before visualizing and taking photos under the microscope. Images of IHC were quantified using Image-Pro Plus 6.0 and assessed based on the sum value of integrated optical density (IOD, Area × Intensity) (32 (link), 33 (link)). The average numbers of IOD were counted from three randomly chosen fields.

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Yue S.R., Tan Y.Y., Zhang L., Zhang B.J., Jiang F.Y., Ji G., Liu B.C, & Wang R.R. (2022). Gynostemma pentaphyllum polysaccharides ameliorate non-alcoholic steatohepatitis in mice associated with gut microbiota and the TLR2/NLRP3 pathway. Frontiers in Endocrinology, 13, 885039.

Publication 2022

NLRP3 Caspase-1 IL-1β HRP-conjugated Goat Anti-Rabbit IgG(H+L) DAB

Anti igg Antibodies Antibody Antigen Caspase 1 Chromogen Embedded paraffin Goat Il 1β Liver Microscope Paraffin Peroxidase Rabbit Sodium citrate Tissue Tlr2

Corresponding Organization : Longhua Hospital Shanghai University of Traditional Chinese Medicine

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Variable analysis

independent variables

Sodium citrate treatment for antigen retrieval
Primary antibodies used (TLR2, NLRP3, ASC, Caspase-1, IL-1β)
Secondary antibody HRP-conjugated Goat Anti-Rabbit IgG(H+L)

dependent variables

Expression levels of TLR2, NLRP3, ASC, Caspase-1, IL-1β proteins measured by integrated optical density (IOD)

control variables

Liver tissue samples
Paraffin embedding and slide preparation
DAB chromogen for visualization
Microscopic imaging and quantification using Image-Pro Plus 6.0

controls

No positive or negative controls were explicitly mentioned in the protocol.

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