Sp5 confocal multiphoton microscope

Confocal images were obtained using a Leica SP5 confocal/multi-photon microscope in the Imaging Core of the Lerner Research Institute. Retinal whole mounts were imaged with ×25 and ×40 objectives and a step size of 2–3 µm for vasculature and 0.5–1.0 µm for in vivo microglia. In vitro cell were imaged with ×63 objective and a step size of 0.5–1.0 µm. The images were processed and staining intensity was quantified using Volocity software and Image J. The 3-D images were prepared by merging the confocal stacks using Volocity. The retinal loop area, junctions/field, and microglia numbers were quantitated using FIJI Image J software. The distances of pMLC and phalloidin staining from the cell membrane were analyzed using ImagePro software.
Cell polarity was measured using Image J. The following steps were involved for in vivo analysis: (i) image processing to separate microglial cells from background; (ii) construction of a skeleton to represent the spatial structure of cell bodies and branched processes if required; (iii) generation of dendritic tree area to identify the longest and widest axis; (iv) measurement of microglial length in pixels, including cell body and the branches that are along the longest dendritic tree axis. Similarly, the width was measured along the widest axis: (v) division of length by width to get the ratio, i.e., polarity.

Alexa Fluor 488 or 594 conjugated secondary antibodies (Thermo Fisher Scientific, MA, US) were used for 3D culture immunofluorescence assays. MCF10A-PI3Kα^H1047R 3D-acini were fixed (4% PFA) at day 15 and processed for immunofluorescence microscopy analysis as established previously [59 (link)]. Confocal analyses were performed using the Leica SP5 Multi-photon confocal microscope equipped with UV diode (405 nm), argon (458, 476,488 and 514 nm), HeNe (543 nm) and a HeNe (633 nm) lasers. All images were obtained with a x63 objective. Quantitative measurements of optical density were performed with ImageJ (National Institutes of Health, US). Acinar size was calculated with LAS X software (Leica) following the equation [(length × width²)/2 = acini volume (mm³)] and plotted with GraphPad Prism.

Cells were grown on glass cover slips and washed with phosphate-buffered saline (PBS) after siRNA treatment. Next, cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Cells were washed with PBS and blocked in 1% bovine serum albumin-PBS. Pan-Ras and myc (9E10) antibodies (Millipore, Billerica, MA, USA) were used at a 1:100 dilution and Alexa 488-conjugated or Alexa 594-conjugated secondary antibodies (Invitrogen) were used for visualization. 4',6-diamidino-2-phenylindole (Sigma) 0.5 mg/ml was used as a nuclear stain. Confocal analysis was performed using the Leica SP5 Multi-photon Confocal Microscope equipped with UV Diode (405 nm), argon (458, 476, 488 and 514 nm), HeNe (543 nm) and a HeNe (633 nm) lasers.