Immunohistochemical Analysis of ER Stress Chaperons

Hearts were excised from mice, embedded in paraffin, sectioned into 4 μm slices, and stained with H&E^{56 (link)}. To determine the expression level of ER stress chaperons, immunohistochemical staining was performed on paraffin-embedded sections with a primary antibody against the KDEL receptor (1:50 dilution), and then with a biotinylated secondary antibody (undiluted goat anti-rabbit/mouse (H+L), Dako Denmark A/S, Hovedstaden, Denmark)^{56 (link)}. The sections were then treated with peroxidase-conjugated biotin–avid in complex using VECTASTAIN ABC-AP and visualized by DAB (3, 3-diaminobenzidine).

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Yao Y., Lu Q., Hu Z., Yu Y., Chen Q, & Wang Q.K. (2017). A non-canonical pathway regulates ER stress signaling and blocks ER stress-induced apoptosis and heart failure. Nature Communications, 8, 133.

Publication 2017

ABC-AP

Antibody Biotin Dilution Embedded paraffin Goat Hearts Mouse Peroxidase Rabbit Receptor antibody

Corresponding Organization :

Other organizations : Huazhong University of Science and Technology, Cleveland Clinic

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Presence of primary antibody against the KDEL receptor (1:50 dilution)

dependent variables

Expression level of ER stress chaperons

control variables

Mouse hearts embedded in paraffin
Mouse heart sections of 4 μm slices
Staining with H&E
Use of biotinylated secondary antibody (undiluted goat anti-rabbit/mouse (H+L), Dako Denmark A/S, Hovedstaden, Denmark)
Treatment with peroxidase-conjugated biotin–avid in complex using VECTASTAIN ABC-AP
Visualization by DAB (3, 3-diaminobenzidine)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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