Lsm700 upright confocal microscope

Dendritic spine quantification was conducted in the motor and visual cortices of Nlgn3^y/−Cyfip1^+/−, Nlgn3^y/−, Cyfip1^+/− males and their WT littermates, as well as of Nlgn3^+/−Cyfip1^+/−, Nlgn3^+/−, and Cyfip1^+/− females and their WT littermates, all of which also expressed EGFP under the Thy-1 promoter. Mice were anesthetized with Euthatal and perfused with 4% paraformaldehyde in 0.1 m PBS. The entire brain was dissected out and postfixed overnight in the 4% paraformaldehyde in 0.1 m PBS, kept in 30% sucrose solution until saturated and stored at −80°C. The brains were cut coronally into 50 μm sections on a cryostat (Leica Biosystems) and immediately mounted on glass slides. The regions of interest were identified using a mouse brain atlas (Paxinos and Franklin, 2004 ), and Z-stack images spaced 0.5 mm apart were acquired on a Zeiss LSM700 upright confocal microscope (Carl Zeiss), using a 40 water-immersion lens. The images were reconstructed into two dimensions using Z-stack maximum intensity projection in ImageJ (NIH). The images were analyzed by an experimenter blinded to the genotype of the animals. Spines were identified manually and counted on a 20- to 250-μm-long stretch of a dendrite, with a minimum of 24 dendrites from four mice, per condition. Spine density was calculated as the number of spines per 10 μm of a dendrite.

Cells were cultured at 2 × 10⁵ cells/well on collagen-coated cover glasses in 6-well plates, transfected with siRNAs as described above and fixed in 4% PFA. Mouse anti-human vinculin 2 µg/ml (Thermo Fisher Scientific Cat# MA5-11690, RRID : AB_10976821) was used as primary antibody and goat anti-mouse Alexa Fluor-488 1:500 (Abcam Cat# ab150113, RRID : AB_2576208) as secondary antibody. Glasses were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen) and visualized by confocal microscopy using a Zeiss LSM700 upright confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Focal adhesions area at the invasive front was quantified by using Fiji software version 1.52n (Fiji, RRID : SCR_002285) (25 (link)).

3T3-L1 adipocytes were grown and differentiated on 8-well chamber slides (Nunc® Lab-Tek® Chamber Slide™ system). Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then washed with Tris-buffered saline (TBS) pH 7.6 (composition in mM: 100 Trizma base, 15 NaCl). Unspecific binding was blocked with 3% donkey serum and the cells were permeabilized with 0.1% Triton X-100 in TBS for 1 h at room temperature. Primary antibodies made in the rabbit (Alomone Labs, Jerusalem, Israel) directed against ORAI1 (1 : 1000), STIM1 (1 : 500) or TRPC1 (1 : 100) were incubated over night at 4°C in blocking solution. Then, mouse monoclonal anti-Caveolin1 (Abcam, Cambridge, U.K.) antibody (1 : 500) was applied and incubated over night at 4°C. Donkey anti-rabbit Alexa 488 and donkey anti-mouse Alexa 647 secondary antibodies were diluted to 1 : 2000 in blocking solution and incubated for 1 h at room temperature. Nuclei were counterstained with Hoechst stain (Molecular Probes, ThermoFisher, Waltham, MA, U.S.A.). Images were acquired using a Zeiss LSM 700 upright confocal microscope (Carl Zeiss, Oberkochen, Germany) and analysed with the Imaris software (Bitplane, Zurich, Switzerland).

Embryos were fixed in 4% PFA at 4° C overnight followed by PBS washes. Subsequently, embryos were transferred through a 15% and 30% sucrose gradation followed by embedding in Tissue-Tek O.C.T. compound (Thermo scientific, Waltham, MA USA). 10 μM sections were prepared using Leica cryostat. For immune-staining sections were fixed in 4% PFA for 5 min on ice, washed in a 1X PBS, blocked in serum containing 3% goat serum, 3% bovine serum albumin and 0.1% Triton X-100 followed by incubation with primary antibodies overnight at 4° C. Primary antibodies used were GFP (1:200, ab13970), Runx1 (1:200, ab92336), CD41 (1:300, ab33661), CD31 (1:100, 553370, BD Pharmingen), Vegfr2/Flk1 (1:100, sc48161) and VE-cadherin (1:100, sc-6458). Secondary antibodies, anti-rabbit biotin, anti-rat biotin, anti-goat biotin and anti-chicken Alexa Fluor-488 (Life Technology) were used at 1:200 concentrations. For biotinylated antibodies signal was amplified using ABC (Vectastatin) and cy3-Tyramide amplification kit (Perkin Elmer). Images were taken using a Zeiss LSM 700 Upright confocal microscope.

Three to four day old female fly guts were dissected and fixed in 500 µl of 4% paraformaldehyde for 20 minutes at room temperature. For nile Red staining (to stain intracellular lipid droplets), fixed guts were incubated in Nile Red solution (diluted 1∶100 in 1X PBS from a 100 µg/ml stock solution in acetone) [48] (link) for 15 minutes at room temperature and then washed with 1X PBS three times before mounting on a slide in 70% Glycerol. The tissue was imaged with a Zeiss LSM700 upright confocal microscope under the 20X/0.8 NA objective. At least 10 guts per genotype were imaged per experiment.
For Oil Red O staining, fixed guts washed twice with MilliQ water, 100% propylene glycol and the incubated in propylene glycol for 10 minutes. The tissue was then incubated in 0.5% Oil Red O in propylene glycol at 60°c for 30 minutes. 10 ml of Oil Red O staining solution was prepared by mixing 6 ml (0.1% Oil Red O) in isopropanol and 4 mL MilliQ H₂O, which was filtered through a 0.45 um syringe filter. After 30 minutes incubation in Oil Red O solution the gut tissue was washed twice in 85% propylene glycol at RT, thrice in MilliQ water before mounting onto a slide in 70% Glycerol. Samples were imaged with a Leica MZ16F dissecting microscope.