Tcs sp2 aobs spectral confocal microscope

Paraffin-embedded human brain tissue sections (2 men and 1 woman for both LB positive and negative cases) were deparaffinized and hydrated through a series of graded ethanol solutions followed by PBS. The sections were incubated in Citrate buffer (10 mM Trisodium citrate, 0.05% Tween-20, pH6.0) at 90°C for 10 min for antigen retrieval. After washing in PBS, tissues were incubated in PBS-T (0.3% Triton X-100 in PBS) for 10 min. Tissues were blocked in 5% normal goat serum for 30 min and incubated in primary antibody at 4°C overnight. Immunostaining was visualized with biotinylated secondary, followed by avidin-biotin (Vectastain Elite Kit, Vector Labs), and 3,3’-diaminobenzidine reaction as previously described [43 (link)]. For immunofluorescence analysis, immunostaining was visualized with either fluorescent secondary (1:250 dilution; Alexa Fluor 488 or 594, Molecular Probes). For immunofluorescence staining of cultured cells, cells were grown and transfected on chamber slides. After antigen retrieval, cells were stained using same methods as tissue staining. Slides were scanned using an AperioScan Scope CS (40×magnification; AperioTechnologies Inc.) and fluorescence images of representative areas were acquired on EVOS FL Digital Microscope (EMS), Olympus IX81-DSU Confocal Microscope (Olympus), or TCS SP2 AOBS Spectral Confocal Microscope (Leica).

TIVE cells were grown overnight on coverslips at a dilution of 1 x 10⁴ cells per well in a 6-well plate. Nuclei isolated from PEL cells were prepared as described [72 (link)], and fixed with a 1:1 ratio of methanol and acetone for 10 min in a humid chamber at 4 ˚C. The samples were blocked in PBS with 3% BSA for 1 h at room temperature, and then incubated overnight at 4 ˚C with either primary anti-Ago2 antibody or blocking solution (control). After washing, the samples were incubated with Alexa-468 anti-rat secondary antibody for 1 hour at room temperature. The slides were then stored at -20 ˚C and imaged using a LEICA TCS SP2 AOBS Spectral Confocal microscope. The images were analyzed and figures were generated using the freeware Vaa3D [73 (link)]. The movie was generated using Volocity® 6.3.

Whole-culture immunofluorescence was performed following fixing of mammary organoids at stated time points, by application of 4% paraformaldehyde at room temperature for 30 min. Each well was then washed with phosphate-buffered saline (PBS)-Glycine, three times for 10 min, and left overnight at 4 °C in 10% horse serum (Invitrogen) diluted in homemade immunofluorescence buffer (Supplementary Table 4). Primary antibodies, as detailed in Supplementary Table 5, were then applied diluted in immunofluorescence buffer overnight at 4 °C. Following three washes in immunofluorescence buffer, secondary antibodies Alexa Fluor 488 goat anti-mouse IgG (1/500, Invitrogen) and Alexa Fluor 568 goat anti-rabbit IgG (1/500, Invitrogen) were applied overnight at 4 °C. Following three washes in immunofluorescence buffer, Hoechst counterstain was applied for 30 min, and samples images in PBS. Confocal microscopy was performed using a Leica TCS SP2 AOBS spectral confocal microscope, using the 20 × objective. Images were then processed using FIJI software.

Pancreases from each group were excised and processed as described previously [27] (link). Sections were cut at a thickness of 5 µm, dewaxed in xylene, and rehydrated in a graded ethanol series. The sections were incubated in PBS containing 3% BSA for 1 h at room temperature to block non-specific binding and then with the following antibodies in PBS containing 3% BSA overnight at 4°C: anti-glucagon receptor (1∶100, bs-3945R; Bioss, Woburn, MA), anti-insulin (1∶100, I2018; Sigma, Madrid, Spain), anti-glucagon (1∶100, G2654; Sigma, Madrid, Spain), and anti 11b-HSD1 (1∶50, sc-20175; Santa Cruz Biotechnology). After washing, the sections were incubated with the corresponding secondary antibodies (1∶1000, Alexa Fluor; Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature. Hoechst 33342 (Life Technologies, Madrid, Spain) was used for nuclear staining. Images were captured using a Leica TCS SP2 AOBS spectral confocal microscope.