Lakhasly

4.7. Fluorescent images were acquired with a Nikon A1R confocal laser scanning system using a 60X oil-immersion objective (numerical aperture 1.4) and 405 nm, 488 nm, and 561 nm lasers, generating up to 9 z-slices with a pixel resolution of 100 nm and z-step size of 250 nm using a Galvano scanner.RNA In Situ Hybridization RNA in situ hybridization was performed using the RNAscope 2.5 FFPE Brown Detection Kit (Advanced cell diagnostic) with probes specific to LMOD1 (NM_012134.2, probe region 892- 2078, Advanced Cell Diagnostics, cat no. 444141) as previously described . Actin filaments were stained with Alexa Fluor 488-conjugated phalloidin (Acti-stain 488, Cytoskeleton, #PHDG1, 100 nM in PBS).Slides were imaged using a Nikon E400 fluorescence/bright field microscope equipped with a Nikon DXM1200 camera , and the SPOT Advanced digital imaging software (Diagnostic Instruments)Immunofluorescence on Smooth Muscle Cells HITC6 cells grown on glass coverslips were fixed with 4% (v/v) paraformaldehyde, permeabilized with 0.5% (v/v) Triton X-100 in phosphate buffered saline (PBS) and blocked with 5% (v/v) normal goat serum.Coverslips were incubated with leiomodin-1 antibody ( 1:3000) overnight at 4 ?C followed by incubation with goat anti- rabbit IgG conjugated with DyLight 549 (1:400).Maximal intensity projection images were rendered with Nikon NIS-Elements.4.8.

4.7. Immunofluorescence on Smooth Muscle Cells
HITC6 cells grown on glass coverslips were fixed
with 4% (v/v) paraformaldehyde, permeabilized with
0.5% (v/v) Triton X-100 in phosphate buffered saline
(PBS) and blocked with 5% (v/v) normal goat serum.
Coverslips were incubated with leiomodin-1 antibody
( 1:3000)
overnight at 4 °C followed by incubation with goat anti-
rabbit IgG conjugated with DyLight 549 (1:400). Actin filaments were stained with
Alexa Fluor 488-conjugated phalloidin (Acti-stain 488,
Cytoskeleton, #PHDG1, 100 nM in PBS). Cells were
mounted with ProLong Glass anti-fade with NucBlue (ThermoFisher Scientific). Fluorescent images were
acquired with a Nikon A1R confocal laser scanning
system using a 60X oil-immersion objective (numerical
aperture 1.4) and 405 nm, 488 nm, and 561 nm lasers,
generating up to 9 z-slices with a pixel resolution of 100
nm and z-step size of 250 nm using a Galvano scanner.
Maximal intensity projection images were rendered
with Nikon NIS-Elements.
4.8. RNA In Situ Hybridization
RNA in situ hybridization was performed using the
RNAscope 2.5 FFPE Brown Detection Kit (Advanced cell diagnostic) with probes
specific to LMOD1 (NM_012134.2, probe region 892-
2078, Advanced Cell Diagnostics, cat no. 444141) as
previously described . Slides were imaged using a
Nikon E400 fluorescence/bright field microscope
equipped with a Nikon DXM1200 camera , and the SPOT Advanced
digital imaging software (Diagnostic Instruments)