Histological and Immunofluorescence Analysis of Adipose Tissue

Formalin-fixed paraffin-embedded tissues were cut into 5-µm-thick sections. Hematoxylin and eosin staining were conducted following standard protocols, and the stained tissue sections were visualized under a light microscope equipped a Kodak digital camera. Adipocyte size was determined using ImageJ software (NIH).
Immunofluorescence analysis was performed as previously described (Zhou et al., 2016 (link)) using primary antibodies against the following proteins and peptides: Trx2 (Santa Cruz; catalog no. sc-50336), FABP4 (Cell Signaling Technology; catalog no. 3544; and Abcam; catalog no. ab93945), p62/SQSTM1 (Cell Signaling Technology; catalog no. 7695), phospho–NF-κB p65 (Cell Signaling Technology; catalog no. 3033), NF-κB p65 (Cell Signaling Technology; catalog no. 8242), F4/80 (BioLegend; catalog no. 123116), TOMM20 (Abcam; catalog no. ab56783), insulin (Cell Signaling Technology; catalog no. 8138), and glucagon (Abcam; catalog no. ab10988). TUNEL (Roche; catalog no. 11684795910) staining performed according to the manufacturer’s instructions. Fluorescent images were acquired using a fluorescence microscope (Zeiss).

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He F., Huang Y., Song Z., Zhou H.J., Zhang H., Perry R.J., Shulman G.I, & Min W. (2020). Mitophagy-mediated adipose inflammation contributes to type 2 diabetes with hepatic insulin resistance. The Journal of Experimental Medicine, 218(3), e20201416.

Publication 2020

digital camera FABP4 p62/SQSTM1 phospho–NF-κB p65 NF-κB p65 F4/80 TOMM20 insulin fluorescence microscope

Adipocyte Antibodies Camera Eosin Fabp4 Fluorescence microscope Formalin Glucagon Hematoxylin Immunofluorescence Insulin Insulin cell Microscope light Nf κb p65 Paraffin Peptides Proteins Stained tissue Tissues Tomm20 Tunel

Corresponding Organization : Yale University

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Variable analysis

independent variables

Tissue preparation (Formalin-fixed paraffin-embedded)
Staining techniques (Hematoxylin and eosin staining, Immunofluorescence analysis)

dependent variables

Adipocyte size
Trx2 expression
FABP4 expression
P62/SQSTM1 expression
Phospho–NF-κB p65 expression
NF-κB p65 expression
F4/80 expression
TOMM20 expression
Insulin expression
Glucagon expression
TUNEL staining

control variables

Standard protocols for Hematoxylin and eosin staining
Manufacturer's instructions for TUNEL staining

positive controls

None specified

negative controls

None specified

Annotations

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