Immunohistochemical Analysis of Mouse Pancreas

Mouse pancreas was fixed in 10% neutral buffered formalin and embedded in paraffin for sectioning. IHC and IF were performed as previously described^{33 (link)}. Antibodies used are listed in Supplementary Table 2.. Some slides, as indicated, also included nuclear staining by DAPI (4’, 6-diamidino-2-phenylindole). Ki67 IHC was used to calculate Ki67⁺ cells against total cell number from >4 views in each mouse. Co-IF of PDX1 and insulin was used to calculate the frequency of PDX1⁺ cells among insulin⁺ cells from 3–5 representative islets for each mouse. Co-IF of cleaved caspase-3 (Asp175) and insulin was used to calculate the frequency of apoptotic cells among insulin⁺ cells from 3–5 representative islets for each mouse. H&E staining was conducted by following the manufacturer’s instructions (ab245880, Abcam). Images were taken using a BZ-X700 microscope (Keyence) and BZ-X viewer software (Keyence, version 1.0.0). Islet area was measured using ImageJ and average islet area was calculated from more than 8 representative islets for each mouse.

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Cao M., Isaac R., Yan W., Ruan X., Jiang L., Wan Y., Wang J., Wang E., Caron C., Neben S., Drygin D., Pizzo D.P., Wu X., Liu X., Chin A.R., Fong M.Y., Gao Z., Guo K., Fadare O., Schwab R.B., Yuan Y., Yost S.E., Mortimer J., Zhong W., Ying W., Bui J.D., Sears D.D., Olefsky J.M, & Wang S.E. (2022). Cancer-cell-secreted Extracellular Vesicles Suppress Insulin Secretion through miR-122 to Impair Systemic Glucose Homeostasis and Contribute to Tumour Growth. Nature cell biology, 24(6), 954-967.

Publication 2022

ab245880 BZ-X700 microscope BZ-X viewer software

Antibodies Apoptotic Caspase 3 Dapi Embedded paraffin Formalin Insulin Insulin cells Microscope Mouse Pancreas Pdx1

Corresponding Organization :

Other organizations : University of California, San Diego, Regulus Therapeutics (United States), City of Hope, University of California, Riverside, Arizona State University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Frequency of PDX1+ cells among insulin+ cells
Frequency of apoptotic cells among insulin+ cells
Ki67+ cells against total cell number
Islet area

control variables

Mouse pancreas was fixed in 10% neutral buffered formalin and embedded in paraffin for sectioning
IHC and IF were performed as previously described (link provided)
Antibodies used are listed in Supplementary Table 2
Some slides included nuclear staining by DAPI

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

Annotations

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