Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.
In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations based on their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells, and predicting assay outcomes using machine learning, among many others. Here we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, golgi apparatus, plasma membrane, endoplasmic reticulum, and mitochondria. The original protocol was updated in 2016 based on several years' experience running it at two sites, after optimizing it by visual stain quality. Here we describe the work of the Joint Undertaking for Morphological Profiling (JUMP) Cell Painting Consortium, aiming to improve upon the assay via quantitative optimization, based on the measured ability of the assay to detect morphological phenotypes and group similar perturbations together. We find that the assay gives very robust outputs despite a variety of changes to the protocol and that two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1 to 2 weeks for a typically sized batch of 20 or fewer plates; feature extraction and data analysis take an additional 1 to 2 weeks.
Highlights d A mature olfactory sensory neuron expresses one odorant receptor gene d The expression areas of 68 odorant receptor genes are classified into 9 zones d These zones are highly overlapping and strikingly complex when viewed in 3D
In the mouse, axons of olfactory sensory neurons (OSNs) that express the same odorant receptor (OR) gene coalesce into one or a few glomeruli in the olfactory bulb. The positions of OR-specific glomeruli are traditionally described as stereotyped. Here, we have assessed quantitatively the positions of OR-specific glomeruli using serial two-photon tomography, an automated method for wholeorgan fluorescence imaging that integrates two-photon microscopy with serial microtome sectioning. Our strategy is multiplexed. By repeated crossing, we generated two strains of mice with genetargeted mutations at four or five OR loci for a total of six ORs: MOR23 (Olfr16), mOR37A (Olfr155), M72 (Olfr160), P2 (Olfr17), MOR256-17 (Olfr15), and MOR28 (Olfr1507). Glomerular imaging relied on intrinsic fluorescence of GFP or DsRed, or on wholemount immunofluorescence with antibodies against GFP, DsRed, or β-gal using the method of immunolabeling-enabled threedimensional imaging of solvent-cleared organs (iDISCO). The highresolution 3D-reconstructed datasets were segmented to identify the labeled glomeruli and to assess glomerular positional variability between the bulbs of one mouse (intraindividual) and among the bulbs of different mice (interindividual). In 26 mice aged 21 or 50 d or 10 wk, we made measurements of the positions of 352 glomeruli. We find that positional variability of glomeruli correlates with the OR: For instance, the medial MOR28 glomerular domain occupies a surface area that is an order of magnitude larger than the surface area of the medial MOR23 glomerular domain. Our results quantify the level of precision that is delivered by the mechanisms of OSN axon wiring, differentially for the various OSN populations expressing distinct OR genes.olfaction | glomerulus | gene targeting | axon guidance | olfactory sensory neuron
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