Confocal lsm700

For immuno-labeling, selected brain sections of LEnt from bregma −2.8 were washed in PBS for 10 mins and then permeabilized in 0.5ml PBS/0.25% Triton X-100 (PBST). Tissues were blocked in blocking solution supplemented with 5% BSA and 5% normal donkey serum in PBST, 1.5-2 hrs at room temperature (RT). Then sections were incubated in primary antibodies diluted in 5% BSA/PBST overnight at 4°C. On the second day, we washed the brain sections 3x in PBST, 15 min each and then incubated the brain sections in 2° antibodies (1:700 for Dylight/Alexa-conjugated antibodies made in donkey purchased from Thermo Fisher Scientific) in 5% BSA/PBST for 2 hrs at RT. For DAPI nuclei stain, DAPI (1:10,000) was diluted in PBST and incubated with brain sections for 15 min followed by brain section washing 2x with PBST then 1x with PBS, 10 min each. The brain sections were mounted onto microscope glass slides in Prolong gold antifade reagent. Images were captured by Carl Zeiss confocal LSM700. Primary antibodies used for brain tissue labeling were as follows: Mouse monoclonal anti-TOMA2, 1:300 (provided by Rakez Kayed); Rabbit Polyclonal anti-HNRNPA2B1, 1:300 (Thermo Fisher Scientific, Cat# PA534939, RRID:AB_2552288); Rabbit Polyclonal anti-LBR, 1:300 (Proteintech, Cat# 12398-1-AP, RRID:AB_2138334); Rabbit Polyclonal anti-TDP43, 1:300 (Proteintech, Cat# 12892-1-AP, RRID:AB_2200505).

Conditioned CDCs were grown on Nunc Lab-Tek^® four-well chamber slides precoated with 10 μg/ml fibronectin and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min on ice. Fixed cells were blocked with 10% donkey serum (Biosera) in 0.1% PBS-Tween [0.9% NaCl, 10 mM Tris base, and 0.05% Tween (all Sigma-Aldrich)] for 1 h at room temperature and then incubated overnight, at 4°C in a humidified chamber, with the primary antibodies anti-cardiac troponin I (cTnI) (1:1,000; Abcam), anti-α-sarcomeric actin (1:1,000; Abcam), anti-myosin heavy chain (1:1,000; Abcam), anti-c-Kit (1:1,000; Santa Cruz), anti-CD90 (1:1,000; BD Pharmingen) anti-Oct-4 (1:1,000; Santa Cruz), anti-Klf-4 (1:1,000; Abcam), anti-Nanog (1:1,000; Abcam), or anti-Sox 2 (1:1,000; Santa Cruz) diluted in PBS in the ratio 1:100. Cells were then incubated with the appropriate secondary antibody conjugated to FITC and the immunofluorescence detected using a confocal microscope (Zeiss Confocal LSM 700). For confocal cytometry, the number of positive cells for each protein was counted in 10 representative fields.