Mz125 microscope

Workers and queens (and one male) belonging to 22 Strumigenys species were collected from their natural nests, representing both short- and long-mandibulate species (Table 1). The anterior parts of the heads were cut off under a Leica MZ 12.5 microscope (Leica, Wetzlar, Germany) and fixed in 2% cold glutaraldehyde in a 50 mM Na-cacodylate buffer at pH 7.3 for 12 h, followed by postfixation in 2% osmium tetroxide in the same buffer for one hour. After dehydration in a graded acetone series, the tissues were embedded in araldite. Serial 1 μm semithin sections were made using a diamond knife with a Leica EM UC6 ultramicrotome (Leica, Wetzlar, Germany). Sections for light microscopy were stained with methylene blue and thionin and observed under an Olympus BX-51 microscope (Olympus, Tokyo, Japan). Double-stained 70 nm thin sections were studied under a Zeiss EM900 electron microscope (Zeiss, Oberkochen, Germany). Samples for the scanning microscopy study were put on aluminium stubs with double-adhesive tape, coated with palladium and viewed under a JEOL JSM-6360 scanning microscope (JEOL Ltd., Tokyo, Japan).

After sacrificing the mother by cervical dislocation, embryos were removed and dissected in phosphate-buffered saline (PBS) under a light microscope. The total number of embryos in each litter was noted, as well as the presence or absence of embryos with gross edema. Each embryo was staged according to its phenotypic appearance, and the head was removed and the thoracic cavity opened. Embryos were scored according to lung lobation, heart apex position, heart outflow tract patterning, and stomach position. Any other gross abnormalities were also noted. All imaging was performed in PBS using a Teledyne Lumenera Infinity3-6URC camera on a Leica MZ12.5 microscope. Tail clips were taken after imaging for genotyping purposes.

Intrinsic optical imaging was performed through the cranial window as previously described (O'Connor et al., 2010 (link)). Mice were lightly anesthetized with isofluorane (0.5% ) and were placed on a heat blanket with their bodies maintained at 37°. Images were acquired using a CCD camera (Retiga-2000RV, Qimaging, Surrey, BC, Canada) through a Leica MZ12.5 microscope (field of view 4.8 x 3.6 mm) with 630 nm LED illumination (Philips LumiLEDs, Amsterdam, Netherlands). The targeted whisker was placed inside a glass pipette connected to a piezoelectric bimorph. The whisker was deflected at 10 Hz for 4 s every 20 s for 10 min. Images were averaged during the 4 s stimulation epoch. A baseline image of the average of the 10 s period proceeding each stimulation epoch was subtracted of this image to generate the intrinsic signal image. Barrels were visible as regions showing decreased 630 nm reflectance. An image of the vasculature was taken with 530 nm LED illumination (Philips LumiLEDs) as a reference for alignment.

The protocol for mammosphere assay was performed using a published method [28 (link)]. The numbers of secondary mammospheres in each well were counted using a Leica MZ12.5 microscope.

Directed in vivo angiogenesis assay was performed using growth factor reduced basement membrane extract. Briefly silicon tubes of 1.5 cm length were closed at one end and prefilled with extract with and without growth factors (FGF2 and FGF21) and allowed to solidify at 37°C for 1 hour. Silicon tubes were implanted subcutaneously in mice. After two weeks, mice were sacrificed and tubes were recovered and imaged using a Leica MZ125 microscope. The extract was further analyzed by hemoglobin quantitation and harvested by paraffin embedding for H&E staining.

Fully engorged female ticks were collected, weighed and put in individual wells in 24-well plates at 26 ^oC. After 14 days of incubation, female ticks began oviposition. After completion of oviposition egg masses from individual female ticks were weighed and placed in individual vials. The embryonic development rate was determined by the percentage of eggs with an embryo per female. Effects derived from the knockdown of the VgR were estimated by measuring egg diameter, size and viability using a Leica MZ12.5 microscope. Pictures were taken by SPOT Insight 11.2 Color Mosaic (Diagnostic Instruments, Inc.) and analyzed by SPOT 5.2 Software (Diagnostic Instruments, Inc.).