Dig dutp

Three different nucleotide probes of AFAP1-AS1 labeled with DIG-dUTP at both of the 3′ and 5′ ends (Invitrogen, Carlsbad, California, USA) were used for ISH experiment in lung cancer specimens. After paraffin sections were dewaxed, hydrated and blocked with endogenous peroxidase, the ISH kit (Boster, Wuhan, China) was used to detect AFAP1-AS1 expression in lung cancer tissues.
The ISH results were scored according to the staining area and depth. The scoring criteria of staining area were as follows: when the number of positive cells was 0, then the score assigned was 0; 5% < positive cells < 25% were scored 1; 25% < positive cells < 50% were scored 2; 50% < positive cells < 75% were scored 3; positive cells >75% were scored 4. The following is the scoring criteria of color depth. Zero points were assigned when the tissue was not colored. One point is assigned when the tissue appeared light yellow. When the tissue was colored light brown, then 2 points were assigned. When the tissue was stained dark brown, then 3 points were assigned. The two scores were multiplied to get the final score of in situ hybridization for each tissue. Finally, the scores less than 4 were judged as low expression of AFAP1-AS1, while the score greater or equal to 4 was assessed as high expression.

In situ hybridization was performed to detect the expression of AFAP1-AS1 in tissue specimens using three 30-nucleotide probes from different regions of AFAP1-AS1. GAPDH was used as a positive control. The probe sequences were as follows.
AFAP1-AS1 probes:
Probe 1: 5′- ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3′,
Probe 2: 5′-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA -3′,
Probe 3: 5′-CCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3′
GAPDH probes:
Probe 1: 5′-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3′
Probe 2: 5′-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3′
Probe 3: 5′-GTCAGAG GAGACCACCTGGTG CTCAGTGTA-3′
The probes were synthesized and labeled with DIG-dUTP at the 3′ end using a kit from Invitrogen (Shanghai, China) [82 (link)–84 (link)]. The in situ hybridization results were independently scored manually by two pathologists who counted 20 sequential high-power fields judged to be representative of the tumor, while remaining blinded to clinical information.

TUNEL staining was carried out as described [57 (link)]. Morpholinos-injected albinos embryos fixed in MEMFA were rehydrated in PBT and washed in TdT buffer (Invitrogen) for 30 min. End labelling was carried out overnight at room temperature in TdT buffer containing 0.5 μm DIG-dUTP and 150 U/ml TdT (Invitrogen). Embryos were then washed for 2 h at 65°C in PBS/1 mM EDTA. DIG was detected with anti-DIG Fab fragments conjugated to alkaline phosphatase (Roche, Indianapolis, Indiana, USA; 1:2,000) and the chromogenic reaction performed using BM purple (Roche, Indianapolis, Indiana, USA). Injection of a Morpholino against Sf3b4 was used as a positive control for induction of cell death [27 (link)]. For each MO, a subset of injected embryos injected was processed for in situ hybridization against sox10 as internal control for the effect of the MOs. TUNEL dots were counted in the neural crest region on each side. The neural crest region was defined as the lateral part of the anterior neural fold. Differences between injected and uninjected sides were plotted as well as the frequency distribution of TUNEL dots on the injected side for each condition.

Target genes (18S rRNA, U2 snRNA-5S, histone H3, and uncharacterized gene FP-9X) were amplified by PCR using the Emerald PCR master mix (Takara, Japan). The primer sets used are shown in Table 1. PCR was performed in a thermal cycler (WK-0518, Wako, Osaka, Japan) under the following conditions: initial denaturation for 2 min at 98 °C, followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min. PCR products were ligated into the pGEM-T Easy Vector (Promega, Madison, USA) and 30 ng of the ligation products were used to transform competent cells (JM109, pGMT-T Easy-Vector Systems, Promega). Transformed cells were plated onto Luria broth (LB) plates containing 100 μg/ml ampicillin, 40 μg/ml 5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside (X-Gal) and 0.05 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG). Isolated colonies were screened by FISH. Probes using FISH screening were prepared by random prime labeling with digoxigenin-12-dUTP (DIG-dUTP) or cyanine-3-dUTP (Cy3-dUTP) in accordance with the kit protocol (Invitrogen, Tokyo, Japan). Then, FISH-positive clones were later transferred into 15 ml test tubes containing 1.5 ml of LB/ampicillin medium and grown at 37 °C overnight. Plasmids from the resulting clones were extracted according to the manufacturer’s protocol using a Mini Plus Plasmid DNA Extraction System (Viogene, NACALAI TESQUE, INC., Kyoto, Japan).