Tcs sp laser scanning confocal microscope

In situ hybridization with ovary samples was performed as described [32 (link)]. Fluorescent RNA probes for osk and GFP were synthesized using the DIG RNA labeling mix (Roche). Samples were mounted on slides with Vectasheild Mounting Medium (Vector Labs), and imaged with the Leica TCS-SP laser scanning confocal microscope.
For RNase protection assays, RNA was isolated from 3–4 day old females using Tri Reagent-LS (Molecular Research Center) as per the manufacturers instructions followed by phenol/chloroform extraction. Assays were performed using the RPA III Kit (Ambion). Following electrophoresis of products in denaturing gels, signals were detected by phosphorimaging with the Typhoon laser scanner (GE Healthcare) and quantitated using Image J. At least three assays were performed for each transgene.

Approximately 5 × 10⁴ MSCs were seeded on sterile coverslips in 12-well plates with incubation overnight at 37 °C in a humidified atmosphere containing 5.0% CO₂. Cells were then statically infected with Ctr D at MOI of 1–4 for 72 h. Infected and uninfected cells were fixed with 4% PFA for 30 min. The fixed cells were washed 3 times with PBS, permeabilized in a blocking buffer with 0.03% (w/v) Triton 100X and finally blocked using the blocking buffer [0.3% bovine serum albumin (BSA) in PBS]; permeabilization and blocking were done for 30 min each at RT. Cells were incubated with the primary antibodies diluted in 0.3% BSA for 60 min at RT against p27 (1:100, mouse, clone: G173–524, BD Pharmingen), C. trachomatis lipopolysaccharide (1:5000, Clone: CF6J12, Abcam Cambridge, UK) and the white DNA staining DAPI-127 (Sigma-Aldrich, Germany). Primary antibody-labeled cells were washed with PBS and treated for 60 min at RT with secondary fluorescent anti-rabbit Cy3 labeled (Red) (Goat, 1:100, Dianova) and Anti-mouse Cy2 labeled (green) (Goat, 1:100, Dianova) Abs diluted in 1% BSA. β-actin was stained with the red color dye phalloidin. The preparation was washed 3 times with PBS for 5 min each at RT. All samples were mounted onto glass slides using Mowiol and examined by a Leica TCS-SP laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).