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Comprehensive Liver Histopathology and Immunofluorescence Analysis

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All stainings were performed on 10 μm fresh-frozen sections. Oil red O (ORO) staining as well as hematoxylin and eosin (HE) staining of liver sections was performed to define liver histopathologies. Immunoflorescent (IF) stainings of mouse liver sections were performed using the following primary antibodies: anti-mouse/human ApoE (ab52607; Abcam); anti-mouse C1q (ab182451; Abcam); anti-mouse CLEC4F (ab2608299; Invitrogen); anti-mouse CD68 (FA11; Serotec); anti-mouse CD31 (ab553370; BD PharMingen); anti-human C3 (A213; ComplementTech); anti-mouse C4 (HM1046; Hycult Biotech); anti-mouse C5 (ab11898, Abcam). We had previously established the specificity of IF microscopy in mouse and human tissues including no primary antibody negative controls, isotype antibody controls, ApoE knockout mouse tissue (i.e., anti-ApoE antibodies) (1 (link)). IF stainings of human sections were performed using the following antibodies: anti-mouse/human ApoE (ab52607; Abcam); anti-human C1q (ab71089; Abcam); anti-human CD68 (EMB11; DAKO); anti-human C5 (A220; ComplementTech). Hepatitis sections as well as the appropriate control tissues were fixated with Delaunay solution prior to IF staining. Stained sections were analyzed using a Leica confocal microscope (SP8, Leica, Germany) using Leica Application Suite (Leica) and ImageJ software.

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Habenicht L.K., Wang Z., Zhang X., Li Y., Mogler C., Huspenina J.S., Schmid R.M., Weber C., Mohanta S.K., Ma Z, & Yin C. (2022). The C1q-ApoE complex: A new hallmark pathology of viral hepatitis and nonalcoholic fatty liver disease. Frontiers in Immunology, 13, 970938.

Publication 2022

ab52607 ab182451 ab71089 Leica confocal microscope Leica Application Suite

Anti antibodies Antibodies Antibody Apoe Confocal microscope Eosin Frozen sections Hematoxylin Hepatitis Human Isotype Knockout mouse Liver Microscopy Mouse Oil red o Tissues

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Variable analysis

independent variables

Primary antibodies used for immunofluorescent (IF) staining
Tissue types (mouse liver, human liver)

dependent variables

Liver histopathologies
Localization and expression of ApoE, C1q, CLEC4F, CD68, CD31, C3, C4, C5 proteins in mouse and human liver sections

control variables

Section thickness (10 μm)
Section type (fresh-frozen)
Staining techniques (Oil red O, hematoxylin and eosin, immunofluorescent)
Microscopy (Leica confocal microscope SP8, Leica Application Suite, ImageJ software)

positive controls

Previously established specificity of IF microscopy in mouse and human tissues
Isotype antibody controls
ApoE knockout mouse tissue

negative controls

No primary antibody negative controls

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