Tsa fluorescein system

Four-micrometer formalin-fixed, paraffin-embedded sections were de-paraffinized with xylene. Samples were hydrated through a descending alcohol series and endogenous peroxidases were inactivated by incubation in 3% H₂O₂ in methanol for 20 min. Antigen unmasking was performed by boiling the tissue sections in 10 mM citric buffer (pH6; for PI(4,5)P₂ and H3K4me3 detection) for 3 min in a microwave followed by 15 min resting at RT. Blocking of unspecific antigen sites was achieved with 50% goat serum (Thermo Scientific, Schwerte, Germany) in PBS for 1h at RT. Incubation with primary antibody against PI(4,5)P₂ was done in a dilution of 1:250 in 2% goat serum over night at 4° C. Detection of PI(4,5)P₂ antibodies was achieved by incubating the sections with a HRP-conjugated goat-anti-mouse-IgM antibody (Santa Cruz) diluted 1:1,000 in 2% goat serum for 1h at RT. The fluorescence signals were generated with the ‘‘TSA™, Fluorescein System’’ (Perkin Elmer, Rodgau, Germany). After the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and embedded in Immunomount (Fisher Scientific, Schwerte) the specific signals were visualized by immunofluorescence microscopy using the Leica microscope DMI6000B, Leica camera DFC365FX and Leica Application Suite software v3.3.0.16799.

Immunofluorescence experiments were performed according to previously published procedures with minor modifications (Sreepathi and Ferraguti, 2012 (link)). Primary antibodies (Table 1) were prepared in 2% NGS and 0.1%Triton X-100 in TBS (TBS-T). After incubation with primary and secondary antibodies (Table 2), the fluorescence signal for VIP was enhanced using a tyramide signal amplification kit (TSA Fluorescein System, PerkinElmer). For the enhancement, sections were first incubated in streptavidin-HRP (diluted 1:500 in 2% NGS in TBS-T) for 30 min at room temperature and then, after two washes in TBS, in a fluorophore tyramide solution for 6 min. Sections were then mounted onto gelatin-coated slides and coverslipped with Vectashield (Vector Laboratories). When mouse monoclonal antibodies were used, sections were pretreated with a Mouse-on-Mouse kit (Vector Laboratories) to reduce endogenous mouse Ig staining.

Digoxigenin (DIG)-labeled antisense RNA probes were transcribed from template RNAs using an appropriate RNA polymerase (T7 polymerase, TaKaRa or SP6 polymerase, Promega) and a DIG RNA labeling mix (Roche). Embryos were fixed in 4% (w/v) PFA in MOPS buffer (0.1 M MOPS, 0.5 M NaCl, and 0.1% [v/v] Tween 20). After several washes in MOPS buffer, the solution was replaced with hybridization buffer (6× SSC, 50% [v/v] formamide, 0.1% [v/v] Tween 20, 5× Denhardt’s solution, and 100 μg/ml yeast tRNA) with 0.1–1 ng/ml of the antisense RNA probe. The probes were hybridized at 50–60°C for 1 week. After hybridization, the probes were washed off in a series of SSC buffers (4× SSC, 2× SSC, or 1× SSC with 50% [v/v] formamide and 0.1% [v/v] Tween 20) at the hybridization temperature; the embryos were next washed with MOPS buffer. To detect alkaline phosphatase (AP), samples were treated with AP-conjugated anti-DIG antibody (Roche) at 4°C overnight and washed several times in MOPS buffer. Finally, AP activity was detected upon addition of NBT/BCIP solution (Roche). A peroxidase-conjugated anti-DIG antibody (Roche) and the TSA fluorescein system (Perkin Elmer) were used to detect fluorescence.

Spleens were embedded in Tissue-Tek OCT compound (Sakura Finetek) and cut into 5μm sections on a Leica CM3050 cryomicrotome (Leica Microsystems). Sections were fixed with 1:1 acetone/methanol and huCD2-biotin was amplified using fluorescein-tyramide according to the TSA Fluorescein System protocol (PerkinElmer). Conjugated antibodies were used at 1:200 followed by 1:2000 dilution of 1mg/mL DAPI (Invitrogen), mounted using Fluoromount-G (SouthernBiotech), and analyzed on the SP5 confocal microscope (Leica).

A DIG-labeled rols probe directed at the transcript encoding the C-terminus of Rols7 was synthesized by in vitro transcription of rols cDNA LD1 [43 (link)] using a DIG-RNA labeling kit (Roche Diagnostics) and following the manufacturer’s instructions. Formaldehyde-fixed embryos were hybridized in situ with the DIG-labeled rols probe essentially as described in Lécuyer et al. [54 (link)]. DIG-labeled rols probes bound to embryos were detected with biotinylated anti-DIG antibody (1:2000; Roche Diagnostics) and the TSA™ Fluorescein System (Perkin Elmer). Fluorescence in situ hybridizations were analyzed by confocal microscopy at various depths within the samples (stacks), which ensured that many ends of the spindle-like FCs were visible in a single picture.