Fluoview 500 confocal microscope

Cutaneous small fiber nerve degeneration was assessed via intraepidermal nerve fiber density (IENFD) profiles. Prior to systemic PBS perfusion, foot pads were collected from the plantar surface of the hind paw, immersed in Newcomer Zamboni’s fixative (Middletown, WI) overnight at 4 °C, cryoprotected overnight at 4 °C in 30% sucrose in 0.1 M sodium phosphate buffer, cryoembedded, sectioned (30 μm) and processed for pan-axonal marker, PGP9.5, immunofluorescence (1:2000 Proteintech, Rosemont, IL). Three images per mouse (3 mm) were collected on an Olympus FluoView 500 confocal microscope using a 20 × 1.2 objective at a resolution of 1024 × 1024 pixels. The optical section thickness was 3.3 μm. Ten images per stack were flattened using max project arithmetic option in MetaMorph (version 7.7.0.00, Molecular Devices). Counts and distances were summed, and the data are presented as the number of fibers per millimeter.

Cells on a poly-lysine-coated cover slip were fixed and permeabilized for 10 min in PBS-containing 4% paraformaldehyde and 0.1% Triton X-100. After blocking for 1 h in PBS with 10% BSA, the cells were incubated with primary (mouse anti-FLAG M2, 1∶1000; anti-FLAG M2, 1∶3000, rabbit anti-V5 or rabbit anti-GFP, 1∶1000 (Invitrogen)) and subsequent secondary antibodies (Alexa-fluor 488–conjugated anti-mouse Ig (1∶1000), or Alexa-fluor 568–conjugated anti-rabbit Ig (1∶1000) (Invitrogen)) in PBS with 1.5% BSA for 1 h at room temperature. The cover slips were mounted on microscope slides. Fluorescence images were obtained with an Olympus Fluoview 500 confocal microscope using 488-nm laser excitation for Alexa-488 or 543-nm for Alexa-568.

TIRF microscopy was performed on a home-built TIRF microscopy system integrated with an Olympus FluoView 500 confocal microscope using an IX-70 base (Olympus Canada) using a high numerical aperture 60× oil-immersion objective (NA = 1.45, Olympus Japan). A thin layer of index-matching oil (n = 1.518) was used to optically couple the objective to the glass surface of the Nunc Lab-Tek II eight well chamber slides (Thermo Scientific). Excitation of eYFP was achieved using an analog modulated 473 nm diode laser (DHOM-L-150 mW, Suzhou Daheng Optics & Fine Mechanics Co, Ltd). Excitation of AF647 was achieved using an analog modulated 643 nm laser (Power Technology, Model LDCU5/A109) with a maximum measured power of 90 mW at the source. A clean-up notch filter (ZET642/20×, Chroma) was used to spectrally clean the excitation. A polarization beam splitter was used to separate between polarized and depolarized emission (PBS251, Thorlabs) (Fig. 8). Fluorescent images are captured using two water-cooled eXcelon-equipped Evolve 512 EMCCD camera (Photometrics) using μ-Manager (version 1.4.19). Under these conditions, we measure an effective pixel size of 127 nm. Imaging for a single fixed cell was performed over a 30 min period, and 5 to 10 cells were imaged. Note that our microscope did not possess a hardware-based perfect focus in place to account for z-drift during the acquisition period.

Individual synthetic peptides (200 µg) labeled with 5(6)-carboxyfluorescein (FAM) were injected into the tail vein of pregnant mice (E11.5-E17.5) and allowed to circulate for 3 h. Following terminal cardiac perfusion with PBS to remove unbound peptide, maternal and fetal tissues were collected for analysis. Organs were snap frozen, or fixed in paraformaldehyde (4% (w/v) in PBS; overnight) and cryoprotected in sucrose solution (30% (w/v) in PBS; 24 h), embedded in OCT (Sakura) and stored at -80 ºC. Tissue sections (8 μm) were fixed in ice-cold methanol (15 min), washed in PBS (2 X 5 min), mounted in Vectashield medium containing DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, USA) and examined on an Olympus Fluoview 500 confocal microscope (Olympus America) or a Zeiss AxioObserver fluorescence microscope (Zeiss, UK). Images were captured at the same exposure so that comparisons of fluorescence intensity could be made between samples.