Understanding the three-dimensional world from two-dimensional immunofluorescent adjacent sections

Abstract

Visualizing tissue structures in three-dimensions (3D) is crucial to understanding normal and pathological phenomena. However, staining and imaging of thick sections and whole mount samples can be challenging. For decades, researchers have serially sectioned large tissues and painstakingly reconstructed the 3D volume. Advances in automation, from sectioning to alignment, now greatly accelerate the process. In addition, immunofluorescent staining methods allow multiple antigens to be simultaneously detected and analyzed volumetrically. The objective was to incorporate multi-channel immunofluorescent staining and automation in 3D reconstruction of serial sections for volumetric analysis. Paraffin-embedded samples were sectioned manually but were processed, stained, imaged and aligned in an automated fashion. Reconstructed stacks were quantitatively analyzed in 3D. By combining automated immunofluorescent staining and tried-and-true methods of reconstructing adjacent sections, we were able to visualize, in detail, not only the geometric structures of the sample but also the presence and interactions of multiple proteins and molecules of interest within their 3D environment. Advances in technology and software algorithms have significantly expedited the 3D reconstruction of serial sections. Automated, multi-antigen immunofluorescent staining will significantly broaden the range and complexity of scientific questions that can be answered with this methodology.

Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization

“…A variety of in situ amplification approaches have been developed, including immunological methods (Macechko et al, 1997;Hughes and Krause, 1998;Kosman et al, 2004), branched DNA methods (Player et al, 2001;Wang et al, 2012;Kishi et al, 2019;Saka et al, 2019), in situ PCR methods (Nuovo et al, 1992;Martínez et al, 1995;Wiedorn et al, 1999) and rolling circle amplification methods (Gusev et al, 2001;Zhou et al, 2001;Larsson et al, 2010). However, for both RNA-ISH (Tautz and Pfeifle, 1989;Harland, 1991;Lehmann and Tautz, 1994;Kerstens et al, 1995;Nieto et al, 1996;Thisse et al, 2004;Piette et al, 2008;Thisse and Thisse, 2008;Wang et al, 2012) and IHC (Takakura et al, 1997;Sillitoe and Hawkes, 2002;Ahnfelt-Ronne et al, 2007;Fujisawa et al, 2015;Staudt et al, 2015), traditional in situ amplification based on enzyme-mediated catalytic reporter deposition (CARD) remains the dominant approach for achieving high signal-to-background in highly autofluorescent samples, including whole-mount vertebrate embryos and FFPE tissue sections. CARD is widely used despite three significant drawbacks: multiplexing is cumbersome due to the lack of orthogonal deposition chemistries, necessitating serial amplification for one target after another (Denkers et al, 2004;Kosman et al, 2004;Clay and Ramakrishnan, 2005;Barroso-Chinea et al, 2007;Tóth and Mezey, 2007;Glass et al, 2009;Stack et al, 2014;Mitchell et al...…”

NVP-BEZ235 (Dactolisib) Has Protective Effects in a Transgenic Mouse Model of Alzheimer’s Disease

“…Thus, although it is possible to speculate that the drug may interfere with microglia activity and neuroinflammatory process, this pharmacological effect should be further investigated. It is equally relevant to point out the limitations of bidimensional (2D) evaluations, such as the lack of volume data and the reduction of information regarding objects in the three-dimensional (3D) structure (Boyce et al, 2010; Fujisawa et al, 2015), which could be further used to corroborate the present histological findings.…”

HCR spectral imaging: 10-plex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent samples

“…However, in whole-mount vertebrate embryos and other challenging imaging settings including thick brain slices, autofluorescence greatly increases the technical challenge of achieving high signal-to-background, motivating the development of in situ amplification methods (Qian et al, 2004;Ramos-Vara and Miller, 2014). For decades, the challenge of achieving high signalto-background in thick autofluorescent samples proved sufficiently daunting that it was predominantly met by using enzyme-mediated catalytic reporter deposition (CARD) for both in situ hybridization (Tautz and Pfeifle, 1989;Harland, 1991;Lehmann and Tautz, 1994;Kerstens et al, 1995;Nieto et al, 1996;Thisse et al, 2004;Piette et al, 2008;Thisse and Thisse, 2008;Wang et al, 2012) and immunohistochemistry (Takakura et al, 1997;Sillitoe and Hawkes, 2002;Ahnfelt-Rønne et al, 2007;Fujisawa et al, 2015;Staudt et al, 2015), which came with unfortunate consequences. Using CARD, multiplexing is cumbersome owing to the lack of orthogonal deposition chemistries, necessitating serial amplification for one target after another (Denkers et al, 2004;Kosman et al, 2004;Clay and Ramakrishnan, 2005;Barroso-Chinea et al, 2007;Tóth and Mezey, 2007;Glass et al, 2009;Mitchell et al, 2014;Stack et al, 2014;Tsujikawa et al, 2017), staining is qualitative rather than quantitative, and spatial resolution is routinely compromised by diffusion of reporter molecules prior to deposition (Tautz and Pfeifle, 1989;Takakura et al, 1997;Sillitoe and Hawkes, 2002;Thisse et al, 2004;Acloque et al, 2008;Weiszmann et al, 2009).…”