880 airyscan confocal microscope

Three days following viral gene transfer, when HSV and GFP expression remained readily detectable, mice were transcardially perfused with 0.1 M sodium phosphate buffer, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were removed and postfixed in 4% PFA overnight at 4 °C. Following postfix, whole brains were stored in 15% sucrose in phosphate buffer with 0.05% sodium azide at 4 °C for 24 h, and then stored in 30% sucrose in phosphate buffer with 0.05% sodium azide at 4 °C until sectioning. Coronal sections at 40μm thick containing the PFC were then prepared on a Lecia VT1000S vibratome in 0.1 PBS. Sections were then mounted and coverslipped using ProLong Gold Antifade. Slides were kept at 4 °C until imaging in a light-blocking slide box until imaging. Sections were imaged and captured with Zeiss 880 Airyscan confocal microscope. Slices were imaged with a 63x oil-immersion magnification.

Stained coverslips were imaged at the UC San Diego Microscopy Core (Grant NS04710) using a Zeiss 880 Airyscan Confocal Microscope. Zen Black Airyscan Image Processing Software version 2.3 SP1 was used to process the images.

CHL cells were transiently transfected with cDNA constructs, as indicated in the figure legends, with Lipofectamine 2000. Twenty-four hours after transfection, cells were fixed with ice-cold 100% methanol for 15 minutes then washed quickly 3 times with Dulbecco’s phosphate-buffered saline (DPBS). Cells were blocked for approximately 1 hour at RT in blocking buffer (90% DPBS, 10% goat serum, and 0.3% Triton X-100). Anti-V5 antibody was diluted 1:1000 in blocking buffer and incubated with cells overnight at RT in a humidified chamber. Cells were washed 3 times for 10 minutes with DPBS. Next, cells were incubated with secondary antibody for 2 hours at RT in a humidified chamber. The secondary antibody, Alexa Fluor 568, was diluted 1:500 in blocking buffer, and cells were washed 3 times for 10 minutes with DPBS and allowed to dry. Cover slips were mounted with ProLong Gold (Invitrogen) with DAPI. Transfected cells were imaged by an investigator blinded to conditions at 63x on a Zeiss 880 AiryScan confocal microscope in the University of Michigan Department of Pharmacology. Images were analyzed by an investigator blinded to condition in ImageJ using Pearson’s correlation coefficient.

We used muIs32[mec-7p::gfp] to visualize FLP cell morphology. To generate synchronous cultures, eggs were collected from gravid hermaphrodites by bleaching with 5% bleach and 5N NaOH in M9 buffer [77 (link)]. Eggs were allowed to hatch overnight in M9, generating a population of L1 arrested larva. Arrested L1 larvae were then transferred to NGM plates seeded with OP50 E. coli. The L1 larvae were allowed to develop at 22°C until the desired stage for imaging. We evaluated the worms after 1, 14, 22, 30, and 40 hours on food to determine FLP morphology at the L1, L2, L3, L4 and young adult stages, respectively. Dauers were selected based on morphology and behavior from recently food-depleted plates. Representative images for each stage were acquired using a Zeiss 880 Airyscan confocal microscope. Twenty animals were observed under an epifluorescence compound microscope at each time point to confirm the consistency of our observations.