Imaging platform and automated fluidics delivery system were similar to those previously described with some modifications. In brief, the flow cell on the sample was first connected to the automated fluidics system. Then the region of interests(ROI) was registered using nuclei signals stained with 10 μg/mL of DAPI (D8417; Sigma). For cell culture experiments, blank images containing beads only were first imaged before the first round of serial hybridization. Each serial hybridization buffer contained three unique sequences with different concentrations of 15-nt readouts conjugated to either Alexa Fluor 647(50 nM), Cy3B(50 nM) or Alexa Fluor 488(100 nM) in EC buffer made from 10% Ethylene Carbonate (E26258; Sigma), 10% Dextran Sulfate (D4911; Sigma), 4X SSC and 1:100 dilution of SUPERase In RNase Inhibitor. The 100 μL of serial hybridization buffers for 80 rounds of seqFISH+ imaging with a repeat for round 1 (in total 81 rounds) were pipetted into a 96 well-plate. During each serial hybridization, the automated sampler will move to the well of the designated hyb buffer and flow the 100 μL hyb solution through a multichannel fluidic valves (EZ1213-820-4; IDEX Health & Science) to the flow cell (required ~25 μL) using a syringe pump (63133–01, Hamilton Company). The serial hyb solution was incubated for 17 minutes for cell culture experiments and 20 minutes for tissue slice experiments at room temperature. After serial hybridization, the sample was washed with ~300 μL of 10% formamide wash buffer (10% formamide and 0.1% Triton X-100 in 2X SSC) to remove excess readout probes and non-specific binding. Then, the sample was rinsed with ~200 μL of 4X SSC supplemented with 1:1000 dilution of SUPERase In RNase Inhibitor before stained with DAPI solution (10 μg/mL of DAPI, 4X SSC, and 1:1000 dilution of SUPERase In RNase Inhibitor) for ~15 seconds. Next, an anti-bleaching buffer solution made of 10% (w/v) glucose, 1:100 diluted catalase (Sigma C3155), 0.5 mg/mL Glucose oxidase (Sigma G2133), 0.02 U/μL SUPERase In RNase Inhibitor, 50 mM pH8 Tris-HCl in 4x SSC was flowed through the samples. Imaging was done with the microscope (Leica, DMi8) equipped with a confocal scanner unit (Yokogawa CSU-W1), a sCMOS camera (Andor Zyla 4.2 Plus), 63 × oil objective lens (Leica 1.40 NA), and a motorized stage (ASI MS2000). Lasers from CNI and filter sets from Semrock were used. Snapshots were acquired with 0.35 μm z steps for two z slices per FOV across 647-nm, 561-nm, 488-nm and 405-nm fluorescent channels. After imaging, stripping buffer made from 55% formamide and 0.1% Triton-X 100 in 2x SSC was flowed through for 1 minute, followed by an incubation time of 1 minute before rinsing with 4X SSC solution. In general, the 15-nt readouts were stripped off within seconds, and a 2-minute wash ensured the removal of any residual signal. The serial hybridization, imaging, and signal extinguishing steps were repeated for 80-rounds. Then, stainings buffer for segmentation purpose consists of 10 μg/mL of DAPI, 50nM LNA T20-Alexa 647, and 1: 100 dilution of Nissl stainings (N21480; Invitrogen) in 1x PBS was flowed in and allowed to incubate for 30 mins at room temperature before imaging. The integration of automated fluidics delivery system and imaging was controlled by a custom written script in Micro-Manager32