Tsa plus fluorescein system

Fluorescence in situ hybridization was performed as previously described using lef1 probes (He et al., 2020 (link)). Briefly, fish with internal organs and hands removed were fixed overnight in 4% paraformaldehyde. kidneys were harvested and embedded in OCT to obtain frozen-sections at 100 μm. Sections permeabilized with proteinase K (10 μg/mL, Roche) in PBT for 20 min with rocking. Digoxigenin-labeled riboprobes were generated from cDNA fragments comprising the sequences of zebrafish lef1 probe (F: 5′-ATGCCGCAGTTGTCAGGTG-3′, R: 5′-CGCTTTCCTCCATTGTTCAGATG-3′). Hybridization was performed as previously described (He et al., 2020 (link)). Anti-DIG POD antibody (11207733910, Roche) and TSA plus fluorescein system (NEL741001KT, PerkinElmer) were used to detect the probe. Fluorescent intensities per unit area were measured using ImageJ.

TSA-FISH with the (TTAGG)_n telomeric probe was performed in A. grossulariata to examine the presence of interstitial telomeric sequences (ITS) in interstitial heterochromatic blocks. The telomeric probe was prepared by nick translation as described above and purified using Sephadex (Illustra Sephadex G-50 fine DNA grade). TSA-FISH was performed as described previously [51 (link)]. Briefly, chromosome preparations after reprobing were treated with 10 mM HCl for 10 min at 37 °C to remove cytoplasm and incubated in 1% hydrogen peroxide for 30 min at room temperature to quench endogenous peroxidase activity. Then the preparations were digested with 100 µg/mL RNase A for 1 h at 37 °C and blocked with 5× Denhardt’s solution for 30 min at 37 °C. Chromosomes were denatured in a probe mix containing 10–30 ng of the labeled telomeric probe, 50% deionized formamide, and 10% dextran sulfate in 2× SSC for 5 min at 70 °C and hybridized overnight. Hybridization signals were enhanced by Antifluorescein-HRP (horseradish peroxidase) conjugate (PerkinElmer, Waltham, MA, USA) diluted 1:1000 and incubated with tyramide solution (TSA Plus Fluorescein system, PerkinElmer) for 5–7 min. The preparations were counterstained and mounted in DABCO-based antifade containing 0.5 µg/mL of DAPI.

Larvae and pupae were injected into the head with 1 μl of 25 mg/ml 5-bromo-2-deoxyuridine (BrdU, Sigma) dissolved in bee saline (130 mM NaCl, 6 mM KCl, 4 mM MgCl₂, 5 mM CaCl₂, 160 mM sucrose, 25 mM glucose, 10 mM HEPES (pH 7.0)) containing 0.01% neutral red, and subsequently kept in an incubator (33 °C) for 20–24 h.
Immediately after dissection, the brains were fixed overnight in 4% paraformaldehyde in PBS at 4 °C. Then, paraffin embedding and sectioning were performed as described above. After that, fluorescence in situ hybridization was performed as described above until the detection step using the TSA Plus Fluorescein System (PerkinElmer).

Neural complexes were surgically removed from the juvenile specimens fixed in 4% paraformaldehyde. The sections were then dehydrated with ethanol and embedded in polyester wax (BDH; Poole, UK). Embedded neural complexes were sectioned at a thickness of 10 μm. Antisense VACHT RNA probes were synthesized using T7-RNA polymerase (Thermo Fisher Scientific, Pittsburgh, PA, USA) with DIG RNA Labeling Mix (Roche). Fluorescein RNA Labeled Mix (Roche) was used to synthesize antisense cionin and CioR1 probes using T7-RNA polymerase. The primer sets used for probe synthesis and probe lengths are listed in Table 1. The wax from the neural complex sections was removed using an ethanol series, and the sections were used for in situ hybridization. Two-color in situ hybridization was performed as previously described^{38 (link)}. First-color staining was performed using the TSA Plus Fluorescein System (PerkinElmer, Waltham, MA, USA) after incubation with anti-fluorescein-HRP (Perkin Elmer). Second-color staining was performed by TSA Plus Cyanine 3 System (PerkinElmer) after incubation with anti-DIG–POD (Roche). The expression signals were observed using an Olympus BX51 microscope (Olympus, Tokyo, Japan).