Avidin biotin blocking kit

For immunohistochemistry (IHC) on murine tissues, lung sections were deparaffinized and rehydrated, and then subjected to heat-mediated antigen retrieval with 10 mM sodium citrate buffer (pH = 6.0) at 95°C for 15 min. After blocking of endogenous peroxidase activity with 3% hydrogen peroxide (15 min, RT), sections were blocked with 10% normal goat serum (1 h, RT) followed by blocking of endogenous biotin using an Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA, United States). Afterwards, primary antibodies for F4/80 (rat anti-mouse F4/80, clone Cl:A3-1, 1:100, AbD Serotec; Kidlington, United Kingdom), and FR-β (rabbit anti-mouse FR-β, 1:400, Genetex, Irvine, CA, United States) were applied on the specimens and incubated overnight at 4°C. Isotype- and concentration-matched IgGs served as negative controls. Next, biotin-labeled goat anti-rat or anti-rabbit secondary antibodies (all from Vector Laboratories) were applied (30 min, RT). This was followed by incubation with the Vectastain ABC Elite HRP kit for 30 min at RT (Vector Laboratories). Finally, stainings were visualized using 3,3′-diaminobenzidine (DAB) in case of F4/80, or 3-amino-9-ethylcarbazole (AEC) (all from Vector Laboratories) in case of FR-β, and sections were counterstained with Mayer's hematoxylin (J.T. Baker, Deventer, Netherlands).

The protocol described in [46 ] was followed. For embryos and newborns, samples were not decalcified. Except for Col2a1, Col10a1, and Ihh (provided by Dr. Licia Selleri), the templates for most riboprobes were generated by PCR from embryonic cDNA, using primers containing the SP6 or T7 RNA polymerase promoters. Sequence of the primers is available upon request. After purification of the PCR product (Qiagen PCR purification kit), DIG-labelled probes were transcribed following manufacturer instructions (Roche), treated with DNAase for 30 min, and purified by LiCl-mediated precipitation in alcoholic solvent. Probes were kept at −80°C in 50% formamide (Fluka). For immunohistochemistry after in situ hybridisation, sections were incubated in citrate buffer (10 mM citric acid, 0.05% Tween 20 [pH 6.0]) for 15 min at 90°C, allowed to cool down, washed with PBSTx, and incubated with 1% H₂O₂ in PBSTx for 1 h to block endogenous peroxidases. After BSA blocking and primary antibody incubation, endogenous biotin was blocked using Avidin/Biotin Blocking kit (Vector #SP-2001), and then the slides were incubated with a biotinylated secondary antibody. A brown precipitate was obtained using a peroxidase-coupled streptavidin-biotin complex (Vectastain Elite ABC Kit, Vector #PK-6100) and DAB substrate (Vector #SK-4100), following manufacturer instructions.

Skin draining LNs (axillary and brachial) were either freshly embedded in tissue freezing medium or fixed overnight in 2% paraformaldehyde/30% sucrose solution at 4°C and embedded in tissue freezing medium. 6–8 μm thick cryostat sections were cut for imaging by the fluorescence microscope. Primary antibodies used included biotinylated or purified anti-B220 (eBiosciences), anti-TCRβ (BD Biosciences), anti-LYVE-1 (Upstate), anti-CD31 (Serotec), anti-Ki67 (Dako), anti-CCL21 (R&D Systems), anti-collagen type IV (Cosmo Bio), FITC-conjugated anti-CD169 (Serotec), biotinylated anti-CD45.1 (eBiosciences) antibodies. Secondary antibodies used included Dylight647-conjugated streptavidin, Cy2 or Cy3-conjugated anti-rat IgG, Dylight647 or Dylight549-conjugated anti-armenian hamster IgG, Dylight 647-conjugated anti-goat IgG and Cy2, Cy3, or Dylight647-conjugated anti-rabbit IgG antibodies (Jackson Immunoresearch). Endogenous avidin and biotin were quenched using the Avidin/Biotin blocking kit (Vector Laboratories).

NIH cells (Fig. 2) were seeded onto cover slips placed in 24-well plates. After 24 h, the medium was changed to new medium containing 360 μM oleic acid. After 20 h, the cells were fixed with 2% formaldehyde for 10 min and stored at 4°C in PBS. For immunolabeling, the cells were treated with Avidin/Biotin Blocking kit (Vector Laboratories), and 1% BSA in PBS for 20 min. The cells were then incubated 2h at room temperature with mouse anti-vinculin (1:100, clone SPM227, Abcam), followed by 30 min biotinylated monovalent donkey anti-mouse IgG (1:200, Jackson ImmunoResearch) and 30 min with Atto425-conjugated streptavidin (1:200, ATTO-TEC). The cells were then incubated for 2h at room temperature with guinea pig anti-ADRP (1:400, Fitzgerald), rabbit anti-mouse syntaxin 5 (1:150, Synaptic System) and rat anti-mouse LAMP1 (1:100, clone 1D4B, Abcam) followed by 30 min incubation with goat anti-guinea pig AF488 (1:400, Life Technologies), goat anti-rabbit Cy3 (1:250, Jackson ImmunoResearch), mouse anti-rat PerCP-eFluor710 (1:100, Affymetrix) and phalloidin-Atto594 (5 μL/mL, Sigma-Aldrich); in the last 5 min, 3 drops of DAPI (2 μg/mL) was added to each well. All incubation steps contained 0.1% saponin and all washing steps contained 0.05% saponin for permeabilization. The cover slips were mounted onto microscope slides with Prolong Gold antifade reagent (Life Technologies).

Freshly isolated thymic lobes were frozen in optimal cutting temperature (OCT) compound (Tissue-Tek) and cryosectioned at a thickness of 10 μm. Tissue sections were fixed with ice-cold acetone for 5 min and blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories) and Protein Block (Dako) according to the manufacturer’s protocol. Tissue sections were then incubated with primary antibodies at 4°C overnight: rabbit anti-mouse CD26 (DPP4) [EPR5883(2), Abcam] and biotin anti-mouse podoplanin (8.1.1, BioLegend). Secondary antibody staining was performed at RT for 30 min with anti-rabbit:AF488 (Invitrogen) and streptavidin-AF555 (Invitrogen). Nuclei were stained with Hoechst 34580 in PBS (according to the manufacturer’s protocol). Sections were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and acquired using an LSM700 confocal microscope (Carl Zeiss AG). Image analysis was performed with ImageJ software (W. S. Rasband, ImageJ, U.S. National Institutes of Health, Bethesda, MD).