Fixation and staining of Drosophila L1 larval brains for immunofluorescence microscopy and preparation for live imaging

2020 ◽  
Author(s):  
Phuong Thao Ly ◽  
John D. Thompson ◽  
Sharyn A. Endow
Keyword(s):  
Immunofluorescence Microscopy ◽  
Cost Effective ◽  
Live Imaging ◽  
Permeable Membrane ◽  
Antibody Staining ◽  
First Instar ◽  
Stem Cell Quiescence ◽  
Cell Quiescence ◽  
3D Printed ◽  
Entire Procedure

Abstract Drosophila first instar (L1) larval brains (LBs) contain frequent quiescent neural stem cells (qNSCs) as well as activated neuroblasts, making them favorable for studying stem cell quiescence and activation. However, the small size of LBs at the L1 stage necessitates the use of modified methods to prepare the LBs for immunofluorescence microscopy (IFM). The protocol described here allows efficient collection of embryos and maturation of larvae to the mid-L1 stage, followed by dissection, fixation and processing of LBs through the antibody staining steps for IFM. The entire procedure can be completed in ~3-5 days. Methods are also described for use in preparing L1 brains for live imaging experiments, including a file to create accessible and cost-effective 3D printed slides that can be fit with an O2-permeable membrane for live imaging.

scholarly journals 3D printed microfluidic lab-on-a-chip device for fiber-based dual beam optical manipulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Haoran Wang ◽  
Anton Enders ◽  
John-Alexander Preuss ◽  
Janina Bahnemann ◽  
Alexander Heisterkamp ◽  
...  
Keyword(s):  
Optical Fibers ◽  
Single Cells ◽  
Lab On A Chip ◽  
Cost Effective ◽  
Optical Manipulation ◽  
Hydrodynamic Focusing ◽  
Trapping Region ◽  
Dual Beam ◽  
3D Printed ◽  
On Chip

Abstract3D printing of microfluidic lab-on-a-chip devices enables rapid prototyping of robust and complex structures. In this work, we designed and fabricated a 3D printed lab-on-a-chip device for fiber-based dual beam optical manipulation. The final 3D printed chip offers three key features, such as (1) an optimized fiber channel design for precise alignment of optical fibers, (2) an optically clear window to visualize the trapping region, and (3) a sample channel which facilitates hydrodynamic focusing of samples. A square zig–zag structure incorporated in the sample channel increases the number of particles at the trapping site and focuses the cells and particles during experiments when operating the chip at low Reynolds number. To evaluate the performance of the device for optical manipulation, we implemented on-chip, fiber-based optical trapping of different-sized microscopic particles and performed trap stiffness measurements. In addition, optical stretching of MCF-7 cells was successfully accomplished for the purpose of studying the effects of a cytochalasin metabolite, pyrichalasin H, on cell elasticity. We observed distinct changes in the deformability of single cells treated with pyrichalasin H compared to untreated cells. These results demonstrate that 3D printed microfluidic lab-on-a-chip devices offer a cost-effective and customizable platform for applications in optical manipulation.

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