A OneStep Solution to Fix and Stain Cells for Correlative Live and Fixed Microscopy

Abstract

Correlating the location of subcellular structures with dynamic cellular behaviors is difficult when working with organisms that lack the molecular genetic tools needed for expressing fluorescent protein fusions. Here, we describe a protocol for fixing, permeabilizing, and staining cells in a single step while imaging on a microscope. In contrast to traditional, multi‐step fixing and staining protocols that take hours, the protocol outlined here achieves satisfactory staining within minutes. This approach takes advantage of well‐characterized small molecules that stain specific subcellular structures, including nuclei, mitochondria, and actin networks. Direct visualization of the entire process allows for rapid optimization of cell fixation and staining, as well as straightforward identification of fixation artifacts. Moreover, live imaging prior to fixation reveals the dynamic history of cellular features, making it particularly useful for model systems without the capacity for expressing fluorescent protein fusions. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Fixing, permeabilizing, and staining mammalian cells in one step on the microscope

A conserved pressure-driven mechanism for regulating cytosolic osmolarity

“…S3A ) was performed live on the microscope using the OneStep fixing and staining protocol. 95 Briefly, ~2X10 4 cells (in 150 μl of M7 media) were seeded into a 96-well glass bottom plate (dot scientific, MGB096-1-2-LG-L), and imaged using DIC. During imaging, cells were treated with 150 μl of 0.2% DMSO or 10 μM LatB (for final concentrations of 0.1% DMSO and 5 μM LatB).…”

“…We verified that DMSO- and Latrunculin-treated cells continued to pump their contractile vacuoles. Then, 150 μl of solution was removed from the well and 150 μl of OneStep fixing and staining solution (100 mM sucrose, 50 mM sodium phosphate buffer, 3.6% PFA, 0.025% NP-40, DAPI, and Alexa Fluor 488 Phalloidin-488: see 95 for additional details) were added. Imaging continued, adding in fluorescence to detect actin polymer and DNA.…”

Data-driven microscopy allows for automated targeted acquisition of relevant data with higher fidelity

“…Such systems are typically tailored for the high-resolution to super-resolution domain, custom-built, and require specialized expertise. Because of the inherent spatial resolution of this domain, such systems are difficult to calibrate and force researchers into using single-system multi-modal imaging (1, 2), with on-stage fixation and labeling protocols (1, 3, 4). On the other hand, multi-system correlative imaging (5, 6, 7, 8, 9) is dependent on finding a transformation between the respective coordinate systems.…”