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(A) Sample and plate preparation steps for mounting before performing the 3D-4i multiplexing protocol. (B) Visualization of the zebrafish embryos mounted inside the imaging plate well. Example fluorescence image in side view (x–z) acquired with a spinning disk confocal microscope and 1 μm z-step size (arrow depicts imaging direction from objective). Scale bar: 50 μm. (C) Example overview of a 96-well imaging plate with 5–6 zebrafish embryos mounted each in 24 individual wells. DAPI signal with color corresponding to cycle 1 (red) and 4 (blue) of the 3D-4i multiplexing protocol. Total number of embryos is 138; the number of detached embryos during a four-cycle 3D-4i protocol is 1 (second column from the left, third row from the top).

Journal: Bio-protocol

Article Title: 3D Iterative Immunofluorescence Imaging on Whole-Mount Samples

doi: 10.21769/BioProtoc.5838

Figure Lengend Snippet: (A) Sample and plate preparation steps for mounting before performing the 3D-4i multiplexing protocol. (B) Visualization of the zebrafish embryos mounted inside the imaging plate well. Example fluorescence image in side view (x–z) acquired with a spinning disk confocal microscope and 1 μm z-step size (arrow depicts imaging direction from objective). Scale bar: 50 μm. (C) Example overview of a 96-well imaging plate with 5–6 zebrafish embryos mounted each in 24 individual wells. DAPI signal with color corresponding to cycle 1 (red) and 4 (blue) of the 3D-4i multiplexing protocol. Total number of embryos is 138; the number of detached embryos during a four-cycle 3D-4i protocol is 1 (second column from the left, third row from the top).

Article Snippet: Laboratory centrifuge with rotors compatible with 96-well plate inserts (e.g., Eppendorf, model: 5810R) 8.

Techniques: Multiplexing, Imaging, Fluorescence, Spinning Disk, Confocal, Microscopy