Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations

Abstract

The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.

Optimization and quantitative evaluation of fluorescence in situ hybridization chain reaction in clarified fresh-frozen brain tissues

“…In the current study, we sought to assess the compatibility and efficiency of HCR FISH in freshfrozen brain samples cleared using different approaches and select the suitable platform to map and analyze transcripts. Based on the factors like clearing capability, target biomolecule fixation, ability to perform ex-vivo labeling, fluorescence retention and imaging depth, we explored only two transparency techniques, CLARITY (Chung et al 2013;) with modifications Tomer et al 2014;Krolewski et al 2018), and iDISCO+ (Renier et al 2014). We chose freshfrozen rodent brains as starting material to optimize steps like RNA fixation, probe permeability, hybridization and tissue clearing conditions in intact samples.…”

“…Moreover, when immunochemistry was performed using secondary antibodies tagged with the same fluorophores in combination with tissue clearing methods, the above mentioned differences in fluorescence output were not observed (Krolewski et al 2018). This further indicates that conjugation chemistry likely plays a significant role in the stability of the fluorophores tagged on to the DNA hairpins vs. antibodies.…”

“…CLARITY sample preparation: Tissues were prepared as described in (Tomer et al 2014;Krolewski et al 2018) with modifications to immersion-fixation and the FISH protocol. After fixation, tissues were rinsed in boric acid buffer (0.2 M borate/0.1% Tween-20, pH 8.5) and then immersed in 2% hydrogel solution (2% acrylamide, 0.25% VA-044 initiator, 1x phosphate buffersaline (PBS), 4% PFA) for 24 h at 4ºC.…”

“…Imaging of large intact samples was performed on the CLARITY-optimized light-sheet microscope (COLM) (Tomer et al 2014;Krolewski et al 2018). RI-matched samples were mounted in a quartz cuvette and filled with 88% histodenz or DBE.…”

“…To date, several tissue clearing approaches have been used to achieve rodent brain transparency but only a few have proved to be versatile and successful across a wide range of applications (Silvestri et al 2016;Azaripour et al 2016;Wan et al 2018). Based on factors like clearing capability, target biomolecule fixation, ability to perform ex-vivo labeling, fluorescence retention and imaging depth, we chose to explore two methods 1) tissue lipid matrix replacementbased CLARITY (Chung et al 2013) with modifications (Tomer et al 2014;Yang et al 2014;Zheng and Rinaman 2016;Krolewski et al 2018) and 2) non-aqueous solvent-based iDISCO+ (Renier et al 2014). There has been pioneering work done (Sylwestrak et al 2016;Kramer et al 2018;Park et al 2018) but the combination of HCR FISH with clearing techniques still remains relatively unexplored especially with fresh-frozen tissues.…”