Tsa fluorescein

Brains were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) overnight, rinsed in PBS the following day, and were subsequently placed in 30% sucrose in PBS overnight. The cryopreserved brains were then placed in Optimal Cutting Temperature (OCT) compound (Sakura Finetek, Torrance, CA) and frozen on dry ice, after which they were sectioned into 12 µm coronal slices. The following primary antibodies were used: rabbit polyclonal anti-IbaI (1:500; Wako Laboratory Chemical, Osaka, Japan), anti-GFAP (1:500; Agilent Technologies, Santa Clara, CA), anti-cleaved caspase-3 (1:500; Cell Signaling Technology, Danvers, MA). Immunohistochemistry was performed as described previously^{91 (link)}. For biocytin detection, sectioned tissue was stained using the ABC-HRP kit (Vector Labs, Burlingame, CA) and TSA Fluorescein (PerkinElmer, Waltham, MA). Sections were nuclear counter-stained with DAPI (Molecular Probes, Eugene, OR).

The conditions and methods for fluorescence in situ hybridization and fluorescence in situ hybridization combined with immunostaining are described in detail by Lécuyer, E. et al.^{12 (link),15 (link),28 (link)}. The following antibodies were used for fluorescence in situ hybridization: Biotin-SP (long spacer) IgG fraction monoclonal mouse anti-Digoxin (1:400; Jackson ImmunoResearch Laboratories Inc.; 200-062-156), Peroxidase IgG fraction monoclonal mouse anti-Biotin (1:100; Jackson ImmunoResearch Laboratories Inc.; 200-032-211), TSA Fluorescein (1:50; PerkinElmer; FP1013), and TSA Reagent Alexa Fluor 594 Tyramide (1:50; Molecular Probes; T20950). The following antibodies were used for immunostaining: rabbit polyclonal anti-anillin IgG (1:500; Provided by Dr. Maria G. Giansanti) (Giansati et al., 2015; Sechi et al., 2017), mouse anti-dlg IgG (1:100; Developmental Studies Hybridoma Bank (DSHB); 4F3), rabbit polyclonal anti-RFP IgG (1:100; MBL; PM005), mouse monoclonal anti-Lamin IgG (1:100; DSHB; ADL67.10) and mouse monoclonal anti-Peanut IgG (1:100; DSHB; 4C9H4). Actin was stained with Alexa Fluor^TM 488 Phalloidin (1:100; Thermo Fisher Scientific; A12379). Samples were inspected with a confocal laser-scanning microscope (Olympus FLUOVIEW FV10i).

Extracted skin lesions were fixed in 4% paraformaldehyde for 24 h at 4°C; the tissues were then immersed overnight in 10% sucrose, 30% sucrose, or OCT compound, followed by embedding in OCT compound. Seven-micrometer-thick sections were prepared and stained with hematoxylin and eosin (H&E), in accordance with the appropriate standard protocol. For immunofluorescence staining, the tissue slices were permeabilized and blocked in 25 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.2% saponin, 10% blocking One (Nacalai Tesque). The mycobacteria were stained with anti-Mycobacteria LAM (ViroStat, 5179) after treatment with fluorochrome-conjugated secondary antibody. To visualize neutrophils and macrophages, the following antibodies were obtained from commercial sources; anti-mouse Ly-6G/Ly-6C (Gr-1) (BioLegend, 108403) and anti-mouse F4/80 (BD, 565409), respectively. The samples were incubated with an amplifying secondary antibody (Simple stain mouse MAX-PO, Nichirei, 414311) after reacting with the primary antibody, then stained using a tyramide-based technique (TSA Fluorescein, PerkinElmer). Images were obtained on the LSM 800 Airyscan confocal microscope (Carl Zeiss). The cell numbers in two randomly selected tissue areas (magnification, x20) were calculated using the ImageJ software.

Extracted skin lesions were fixed in 4% paraformaldehyde for 24 h at 4°C; the tissues were then immersed overnight in 10% sucrose, 30% sucrose, or OCT compound, followed by embedding in OCT compound. Seven-micrometer-thick sections were prepared and stained with hematoxylin and eosin (H&E), in accordance with the appropriate standard protocol. For immunofluorescence staining, the tissue slices were permeabilized and blocked in 25 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.2% saponin, 10% blocking One (Nacalai Tesque). The mycobacteria were stained with anti-Mycobacteria LAM (ViroStat, 5179) after treatment with fluorochrome-conjugated secondary antibody. To visualize neutrophils and macrophages, the following antibodies were obtained from commercial sources; anti-mouse Ly-6G/Ly-6C (Gr-1) (BioLegend, 108403) and anti-mouse F4/80 (BD, 565409), respectively. The samples were incubated with an amplifying secondary antibody (Simple stain mouse MAX-PO, Nichirei, 414311) after reacting with the primary antibody, then stained using a tyramide-based technique (TSA Fluorescein, PerkinElmer). Images were obtained on the LSM 800 Airyscan confocal microscope (Carl Zeiss). The cell numbers in two randomly selected tissue areas (magnification, x20) were calculated using the ImageJ software.

RNA in situ hybridisation was carried out as described in [71 (link)], with the following minor modifications: Proteinase K treatment and post-fixations steps in the original protocol were omitted, and prior to hybridization, the probes were heated to 80 °C for 5 min and immediately put on ice before adding to the pre-hybridization buffer. Fluorescent in situ hybridization was performed following [40 ]. Tyramide Signal Amplification (TSA) was performed with TSA kits from PerkinElmer (TSA Fluorescein and TSA Cyanine). Post hybridisation, nuclear staining was achieved by incubating embryos in 1 μg/ml 4–6-diamidino-2-phenylindol (DAPI) in PBS with 0.1% Tween-20 for 15 min. Embryos were mounted in glycerol on Poly-L-lysine (Sigma-Aldrich) coated coverslips, where the germband tissue attaches making it easier to remove the yolk before imaging. Images were taken with an AxioZoom V16 stereomicroscope (Zeiss) equipped with an Axiocam 506 mono and colour digital camera. Brightness and intensity of the pictures were adjusted in Corel PhotoPaint X5 (CorelDraw).

RNAscope (Advanced Cell Diagnostics-ACD, Hayward, CA, USA) fluorescent-field ISH used to detect hMYC, cd79b, and lat mRNA in fish sections. Procedure performed using the Multiplex-Fluorescent-Detection-Kit-v2 (#323110), according to manufacturer instructions (https://acdbio.com/). RNAscope probes used to specifically detect human MYC (#311761-C2), D. rerio cd79b (#511481) and lat (#507681). Probe labels (PerkinElmer, Waltham, MA, USA) as follows: TSA-Plus-Cyanine-3 (#NEL744001KT) for hMYC (yellow fluorescence), TSA-Plus-Cyanine-5 (#NEL745001KT) for cd79b (red), and TSA-Fluorescein (#NEL701A001KT) for lat (green). Slides imaged and analyzed using an Operetta High-Content Imaging System (PerkinElmer) and Harmony 4.1 software.