Lsm780 confocal microscope

HeLa, H413 and SCC9 cells were grown in a six-well culture plate with coverslips. Adhered HeLa cells were washed with PBS and pre-extracted using PHEM buffer (60 mM PIPES, 20 mM HEPES, 10 mM EGTA, 0.2% Triton X-100 and 4 mM MgSO₄) for 5 min at room temperature (RT). The H413 and SCC9 cells were washed with PBS and pre-extracted with 0.05% Digitonin for 2-5 minutes at RT. Pre-extracted cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with rehydration buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.1% Triton X-100) for another 15 min at RT. The cells were blocked using 5% normal goat serum for 30 min and incubated with corresponding primary antibody (anti-Nup88, 1:200 and anti-Nup62, 1:1000) at 4° C for 12 h. After three PBS washes, 1:800 dilutions of Alexa-fluor conjugated secondary antibody (Alexa Fluor 488, Invitrogen, # A11034, Alexa Fluor 568, Invitrogen, # A11031) was added to the cells and allowed to incubate for an hour at RT. Again, three washes of PBS were given, and nuclei were stained with Hoechst 33342 (Invitrogen, # H1399, 1 mg/ml, 1:5000 dilutions). The coverslips were mounted on slides with VECTASHIELD mounting medium (# H1000), and images were captured on a Zeiss LSM 780 Confocal Microscope and Olympus Confocal Laser Scanning Microscope-FY3000. All images were analyzed using ZEN (Zeiss) or Image J/Fiji software.

Mouse bone specimens were first fixed and then decalcified using 10% EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 14 days with constant shaking. For the histological assays, the detailed protocols were reported in a previous study.⁵⁸ In short, the samples were then dehydrated and embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) or in paraffin. Four-μm-thick coronal-oriented femur sections were prepared for TRAP staining. Forty-μm-thick coronal-oriented femur sections were prepared for IF staining. The detailed protocols were described in a previous study.⁵⁸ Briefly, the sections were incubated with primary antibodies against mouse TLR2 (Santa Cruz Biotechnology, sc-21759, 1:200), Siglec15 (PA5-48221, Thermo Fisher Scientific, 1:100), ST3GAL1 (PA5-21721, Thermo Fisher Scientific, 1:50), and TRAP (Abcam, ab191406, 1:100) for 12 h at 4 °C. For sialic acid detection, biotinylated Maackia Amurensis Lectin II (MAL II) (Vector Laboratories, CA, USA) was used to label the α2,3 linkage, and biotinylated Sambucus Nigra Lectin (SNA) (Vector Laboratories, CA, USA) was used to label the α2,6 linkage. Fluorescein-conjugated streptavidin (Vector Laboratories, CA, USA) was used for the addition of a fluorescent label to biotinylated sialic acid conjugates. A Zeiss LSM 780 confocal microscope and an Olympus BX51 microscope were used for image capture.

Dissected bones were fixed in 4% paraformaldehyde overnight, decalcified in 10% EDTA for four days, and dehydrated in 30% sucrose for two days. Bones were sectioned (10 µm) using the CryoJane tape-transfer system (Leica). Sections were blocked in PBS with 10% horse serum for 30 min and then stained overnight at 4°C with goat IgG control (R and D Systems, 1:500), goat anti-Clec11a antibody (R and D systems, 1:500), rabbit anti-Aggrecan antibody (Chemicon, 1:500), rabbit anti-Perilipin antibody (Sigma, 1:2000) or goat anti-Osteopontin antibody (R and D, 1:500). Donkey anti-goat Alexa Fluor 488, Donkey anti-goat Alexa Fluor 647 and donkey anti-rabbit Alexia Fluor 555 were used as secondary antibodies (Invitrogen, 1:500). Slides were mounted with anti-fade prolong gold with DAPI (Invitrogen). Images were acquired using a Zeiss LSM780 confocal microscope or Olympus IX81 microscope.

All images were captured as Z sections in Zeiss LSM 780 confocal microscope and Olympus Fluoview FV10i (Panel 7). Same settings were used for each set of experiments. All the experiments were repeated at least thrice to ensure reproducibility. Mostly, 10 lymph glands were analyzed per genotype for quantification analysis. Data expressed as mean ± standard deviation of values from three sets of independent experiments. At least 10 images of the lymph gland/niche were analyzed per genotype, and statistical analyses performed employed two-tailed Student’s t-test. p-values of <0.05, <0.01, and <0.001, mentioned as *, **, and ***, respectively, are considered as statistically significant. All quantitative analysis was plotted using GraphPad.