Cxk41 inverted microscope

Monolayer cultures of hMSCs in contact with hydrogels (9 mm², H_c and H₂) were fixed in 4% formalin and immunohistochemistry was performed at 1, 7, and 14 days after exposure, using anti-IGF-I-primary antibody, anti-goat IgG secondary antibody, biotinylated secondary antibodies, and the ABC method with diaminobenzidine as the chromogen. Cells maintained in the presence or absence of free vector and cells cultured with non-loaded hydrogel pieces in duplicate were used as controls. Monolayer pictures were recorded with an Olympus CXK41 inverted microscope (Tokyo, Japan).

Spleens were harvested on day 9 and embedded in OCT (CellPath) and frozen in isopentane. Eight μm slices were air-dried overnight, fixed in 100% acetone and blocked with 2.5% normal goat serum and stained for BCL₁ cells (anti-BCL₁ idiotype), normal B cells (anti-B220), neutrophils (anti-Ly6c/Ly6g), macrophage (anti-F4/80) and monocyte (anti-CD14). Sections were treated with a peroxidase inhibitor (Pierce) for 10 min before incubation with an HRP-conjugated anti-rat IgG polymer for 30 min, followed by 3,3’-diaminobenzidine for 5 min, and counterstained with haematoxylin (all from Vector Laboratories). Images were recorded using a CXK41 inverted microscope equipped with a CC12 color camera, Plan Achromat 4 x 0.25 objective lens and Cell B software (all from Olympus).