# Protocol for volume correlative light X-ray and electron microscopy of endothelial cells in mouse tissue

## TL;DR

This paper introduces a protocol combining light, X-ray, and electron microscopy to study endothelial cells in mouse tissue with high precision.

## Contribution

A novel protocol for ROI targeting in endothelial cells using X-ray μCT and CLEM techniques.

## Key findings

- The protocol uses vasculature patterns and EC nuclei positions for accurate ROI targeting.
- It is suitable for thin or translucent tissues with visible landmarks in both light and X-ray microscopy.
- Open-source programs are used for data correlation and ROI targeting.

## Abstract

Correlative light and electron microscopy (CLEM) greatly facilitate capturing the ultrastructure of spatially and/or temporally rare events. Here, we present a protocol for targeting regions of interests (ROIs) in tissue endothelial cells (ECs) using X-ray micro-computed tomography (μCT). We describe steps for ROI targeting guided by vasculature patterns and positions of EC nuclei visualized by light and X-ray microscopy. The protocol is applicable to thin or translucent tissues that contain defined landmarks visible in both light and X-ray microscopy.

For complete details on the use and execution of this protocol, please refer to Reglero-Real et al.1

•Instructions for in vivo fluorescence labeling of tissue ECs•Procedures for confocal microscopy to achieve successful ROI targeting•Steps for sequential trimming and X-ray μCT to obtain the final block for SBF-SEM•Guide for data correlation and ROI targeting using open-source programs

Instructions for in vivo fluorescence labeling of tissue ECs

Procedures for confocal microscopy to achieve successful ROI targeting

Steps for sequential trimming and X-ray μCT to obtain the final block for SBF-SEM

Guide for data correlation and ROI targeting using open-source programs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Correlative light and electron microscopy (CLEM) greatly facilitate capturing the ultrastructure of spatially and/or temporally rare events. Here, we present a protocol for targeting regions of interests (ROIs) in tissue endothelial cells (ECs) using X-ray micro-computed tomography (μCT). We describe steps for ROI targeting guided by vasculature patterns and positions of EC nuclei visualized by light and X-ray microscopy. The protocol is applicable to thin or translucent tissues that contain defined landmarks visible in both light and X-ray microscopy.