Tissue slides were prepared by tumor touch prep method46 (link). Positively charged glass slides were pressed against the surface of thawed frozen tissues. The slides were then immediately fixed by cold Carnoy’s fixative (3:1 methanol:glacial acetic acid, v/v) for 30 minutes and then air-dried. Slides were denatured in hybridization buffer (Empire Genomics) mixed with EGFR-Chr7 probe (EGFR-CHR07–20-ORGR, Empire Genomics) at 75°C for 5 minutes and then incubated at 37°C overnight. The post-hybridization wash was with 0.4x SSC at 75°C for 3 minutes followed by a second wash with 2x SSC/0.05% Tween20 for 1 minute. The slides were then briefly rinsed by water and air-dried. The VECTASHIELD mounting medium with DAPI (Vector Laboratories) was applied and the coverslip was mounted onto a glass slide. Tissue images were scanned under Leica STED 3X/DLS Confocal with 100x magnification.
Hybridization buffer
Manufactured by Empire Genomics
Hybridization buffer is a solution used in molecular biology techniques, primarily in the hybridization step of DNA microarray analysis. Its core function is to facilitate the specific binding of target DNA or RNA sequences to their complementary probes on the microarray surface, enabling the detection and quantification of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Sted 3x dls confocal, by Leica (3 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (3 mentions)
Egfr chr7 probe egfr chr07 20 orgr, by Empire Genomics (2 mentions)
Hybridizer, by Agilent Technologies (1 mentions)
Histology fish accessory kit, by Agilent Technologies (1 mentions)
Pre treatment solution, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abbott (1 mentions)
7 protocols using hybridization buffer
1
EGFR FISH Tissue Touch Prep Protocol
2
EGFR FISH Tissue Touch Prep Protocol
3
Fluorescence In Situ Hybridization Technique
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Metaphase spreads were equilibrated in 2x SSC (30 mM sodium citrate, 300 mM NaCl, pH 7) for ~5 min. They were dehydrated using successive washes of 75, 85, and 100% ethanol for 2 min each and allowed to dry. FISH probes were diluted in hybridization buffer (Empire Genomics) and added to metaphase spreads on slides, along with 22-mm2 coverslips. Samples were denatured at 70–75 °C for 30 s–2 min. Probe hybridization was performed at 37 °C for around 3 h or overnight in a humid and dark chamber. Samples were washed successively in 0.4x SSC and 2x SSC with 0.1% Tween-20. Samples were incubated with DAPI (0.1 µg ml−1 in 2x SSC) for 10 min, then washed with 2x SSC and briefly rinsed with H2O. Samples were mounted with Prolong Gold, #1.5 coverslips, and sealed with nail polish.
4
Fluorescence In Situ Hybridization Protocol
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Fixed samples on coverslips or slides were equilibrated briefly in 2× SSC buffer. They were then dehydrated in ascending ethanol concentrations of 70, 85 and 100% for approximately 2 min each. FISH probes were diluted in hybridization buffer (Empire Genomics) and added to the sample with the addition of a coverslip or slide. Samples were denatured at 72 °C for 2 min and then hybridized at 37 °C overnight in a humid and dark chamber. Samples were then washed with 0.4× SSC then 2× SSC 0.1% Tween 20 (all washes lasting approximately 2 min). 4,6-Diamidino-2-phenylindole (DAPI) (100 ng ml−1) was applied to samples for 10 min. Samples were then washed again with 2× SSC 0.1% Tween 20 then 2× SSC. Samples were briefly washed in double-distilled H2O and mounted with ProLong Gold. Slides were sealed with nail polish.
5
Immunofluorescence Detection of HPV31 in Cells
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Cells were grown on no. 1 glass coverslips and fixed with 4% methanol-free PFA before being permeabilized in PBT. Cells were then blocked with NGS-T and incubated with primary antibody overnight at 4°C in a humidity chamber. Coverslips were washed in PBS (3×) and incubated with secondary antibody. Cells were then treated with ice-cold methanol-acetic acid followed by 2% PFA. Coverslips were treated with RNase-It (Stratagene), dehydrated with 70, 85, and 100% ethanol, and dried for several hours. HPV31 probe (Enzo) in hybridization buffer (Empire Genomics) with Cot1 DNA was added to coverslips, denatured at 75°C, and hybridized overnight at 37°C. Coverslips were washed in wash buffer (0.5× saline-sodium citrate [SSC], 0.1% SDS) followed by a wash in phosphate-buffered detergent. Tyramide signal amplification was performed using TSA kit no. 22 (Life Technologies, Inc.). Cells were counterstained with DAPI and mounted in Gelvatol.
6
FISH Protocol for S100A8 Expression
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Following manufacturer instructions, FISH was carried out using a DAKO Histology FISH accessory Kit (Code K 5799). TMA slides were first heated at 60 °C for 1–2 h before dewaxing and rehydrating. Subsequently, they were boiled for 15 min in a pre-treatment solution (Dako) before cooling to room temperature and washing in Dako buffer (2 × 3 min). The slides were then immersed in pepsin solution (37 °C, 30 min) for protein digestion, washed in Dako buffer (2 × 3 min), dehydrated (2 min each, 70%, 85%, and 96% ethanol), and allowed to air-dry. Then, 3 µL of a custom S100A8 FISH-probe (Empire Genomics) was mixed with 12 µL of hybridization buffer (Empire Genomics) and applied to the slides, which were coverslipped and sealed with coverslip sealant (Dako). They were then placed in a Dako Hybridizer and denatured (83 °C, 3 min) before they were renatured at 37 °C overnight. After this, the slides were rinsed in Stringent Wash Buffer (Dako) (72 °C, 2 min) and Dako wash buffer (2 × 3 min) at room temperature. Next, they were dehydrated (2 min each, 70%, 85%, and 96% ethanol) and dried at 37 °C for 15 min. Finally, 20 µL DAPI (VYSIS Abbott no 06J50-001) was applied to the slides, which were again coverslipped.
7
EGFR Gene Amplification Detection
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For patient tissues, the slightly thawed tissues were transferred to a positively charged glass slide by pressing against the surface of the specimen. The tissue slides were then immediately transferred into Carnoy's fixative (3:1 methanol:glacial acetic acid, v/v), incubated at RT for 30 min and then air-dried. For interphase cell prep, neurospheres were dissociated into single cells and fixed in Carnoy's fixative for 20 min, briefly washed with fixative and resuspended in fixative. Desired amounts of cells were then dropped onto the glass slide and air-dried. A hybridization buffer (Empire Genomics) mixed with EGFR-Chr7 probe (EGFR-CHR07-20-ORGR, Empire Genomics) was applied to the slides and the slides were denatured at 75°C for 5 min. The slides were then immediately transferred and incubated at 37°C overnight. The posthybridization wash was with 0.4x SSC at 75°C for 3 min followed by a second wash with 2x SSC/0.05% Tween20 for 1 min. The slides were then briefly rinsed by water and air-dried. The VECTASHIELD mounting medium with DAPI (Vector Laboratories) was applied and the coverslip was mounted onto a glass slide. Tissue images were scanned under Leica STED 3X/DLS Confocal with 100x magnification. Z-stack acquired at 0.3-0.5 um step size was performed and all analysis conducted based on maximum intensity projection images of the 3D volume of the cells.
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