Slowfade gold antifade reagent with dapi

Dissected tissues were fixed in 4% paraformaldehyde in HBSS, pH 7.6, at 4°C for at least 1 h, rinsed in HBSS and dehydrated in a graded ethanol series, followed by two xylene rinses, paraffin penetration and paraffin embedding. The sections were cut to 10 μm thickness by microtome and “baked” at 60°C overnight onto Superfrost/Plus Microscope glass slides (Fisher). After deparaffinizing and rinsing with PBT (phosphate-buffered saline, PBS, plus 0.1% Tween-20), the sections were placed in 90°C 0.01 M citrate buffer (pH 6.0) for 10 min, for post-fixation antigen recovery, unless otherwise noted. Sections were then treated with blocking buffer as follows: PBT with 10% normal goat serum (NGS), diluted 1:1 with Superblock (Pierce Chemical). Primary antibody was added in HBSS and incubated overnight at 4°C. After rinsing three times with HBSS, sections were incubated with Alexa Fluor 594 secondary antibody (1:2,000, Invitrogen) for 2 h at room temperature. CD36 antibody (R&D Systems MAB25191) was diluted to 2.5 ug/ml. After washing with HBSS twice, sections were mounted in SlowFade Gold antifade reagent with DAPI as the nuclear counterstain (Invitrogen) and coverslipped. Specimens were observed with a Nikon Eclipse E800 fluorescence/DIC microscope.

The method was performed as described in Hadler-Olsen et al. (2010 (link)). In brief, 5-µm-thick sections from ZBF-fixed and paraffin-embedded pin bone tissue (6 h) were heated at 58 °C overnight, deparaffinized in xylene and rehydrated in graded alcohol baths. Two hundred milliliters substrate of dye-quenched (DQ) gelatin (Invitrogen), DQ-collagen or DQ-casein (Life Technologies) diluted 1:50 in reaction buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM CaCl₂, 0.2 mM sodium azide, and pH 7.6) was added to the tissue sections and incubated in dark humidity chamber at 37 °C for 2 h. To evaluate the contribution of proteases, sections were pre-incubated with 0.5 mM GM6001 or 8 mM Pefabloc for 1 h at 37 °C. Sections were then rinsed 2 × 5 min in PBS baths, dipped in Milli-Q water and air-dried for few minutes. The sections were mounted using SlowFade Gold antifade reagent with DAPI (Invitrogen) and examined with a confocal microscope Olympus FluoView FV1000 (Olympus).

Thymus tissue samples were cryosectioned for immunohistochemistry using a Hyrax C60 cryostat (Zeiss). Thymus sections (10 μm) were fixed with 2% (wt/vol) paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4, and acetone, washed in PBS, and blocked with PBS supplemented with 0.1% Triton X-100 and 4% normal goat serum. Subsequently, sections were incubated with the following primary antibodies (diluted in blocking solution) overnight at 4 °C: rat anti-GM-CSF antibody (BD Pharmingen, clone BVD2-21C11, 1:50), mouse anti-CD4 antibody (Biolegend, clone RPA-T4, 1:50) and rabbit anti-CD3 (NOVUS, clone SP7, 1:100). Sections were then washed in PBS and incubated with AF647-labeled goat anti-rat, AF488-labeled goat anti-mouse and AF555-labeled goat anti-rabbit secondary antibodies (Life Technologies, 1:500) overnight at 4 °C. Sections were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen). Fluorescence photomicrographs were captured with a SP5 Leica confocal laser scanning microscope (SP5; Leica) equipped with argon and helium lasers using the 40 × objective lens (oil immersion, NA1.25). Images were processed and merged by Imaris imaging software (Bitplane).

After incubation, the fragments and gels were fixed in 4% paraformaldehyde and used for whole-mount lectin staining. After permeabilization with Triton X-100, the fragments and gels were immersed with Block Ace (DS Pharma Biomedical, Osaka, Japan) and incubated with endothelium-specific lectin [fluorescein griffonia (Bandeiraea) simplicifolia lectin I, isolectin B4; Vector Laboratories, Burlingame, CA, USA]. Cell nuclei were stained with SlowFade Gold antifade reagent with DAPI (Invitrogen, Waltham, CA, USA). Three-dimensional images were obtained using a confocal laser scanning microscope (Olympus, Tokyo, Japan). To evaluate angiogenesis, the number of migrated cells and neovessels and the lengths of neovessels were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The number of migrated cells was calculated by counting cells in the gel. The number of neovessels was calculated by counting lectin-positive vessels that sprouted from the left ventricular fragment toward the gel, and the lengths of neovessels were averaged for all vessel lengths in the gel. The proportion of lectin-positive cells was calculated the positive cell number divided by total cell number.