In situ Quantification of Pancreatic Beta-cell Mass in Mice

Summary

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Abstract

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

Protocol

Part 1: Preparation of Specimens

1. Place a mouse under a dissection microscope, remove the entire pancreas along with the duodenum and spleen still attached, and place the organs on a pre-weighed glass slide. Remove the duodenum by cutting its connective tissue along the side where it is bound to the pancreas. Cut and remove the spleen and excess fat on the pancreas until the pancreas is completely isolated on the slide. (Pancreatic fat is distinguishable by its white color, as differentiated from the slightly darker and tanner color of pancreatic tissue.)
2. Place a coverslip (large cover glass 50x75mm; standard cover glass 25x75mm) over the pancre....

Discussion

The analysis of Virtual Slice images of islets and beta-cell clusters in the intact pancreas provides a large-scale view of the entire distribution of islets. Developments and changes in total islet distribution over time can thus be studied and compared. Such an example is demonstrated in our analysis of pancreatic islet formation (1). A comprehensive analysis of neonatal mice pancreata at various time points (P1-P21) has demonstrated that islets are formed by fission. While beta-cell proliferation fits with a lognormal.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps	Miltex Inc.
Scissors	F.S.T.	14001-13	01n2
Scissors	F.S.T.	14072-10	02s5
Large glass slide	Electron Microscopy Sciences	71862-01	75x51x1.2mm
Large cover glass	Ted Pella, Inc.	260462	50x75mm
Glass Slide	Fisher Scientific	12-550-15	25x75x1.0mm
Cover glass	Fisher Scientific	12-458-5M	75x25mm
Cover glass	Erie Scientific		25mm circle
Fluorescent microscope	Olympus Corporation
Dissection microscope	Olympus Corporation
Stereo Investigator	MBF Bioscience
MIP-GFP mice	Jackson Laboratory